Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/136—Screening for pharmacological compounds

Definitions

• the invention described herein relates to a novel gene and its encoded protein, termed 103P2D6, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express 103P2D6.
• Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, cancer causes the death of well over a half-million people annually, with some 1.4 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
• carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that tlieir lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
• prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 40,000 men die annually of this disease - second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
• PSA serum prostate specific antigen
• the LAPC Los Angeles Prostate Cancer
• SCID severe combined immune deficient mice
• prostate cancer markers include PCTA-1 (Su et al., 1996, Proc.
• PSM prostate-specific membrane
• STEAP Proc Natl Acad Sci U S A. 1999 Dec 7;96(25): 14523-8
• PSCA prostate stem cell antigen
• the present invention relates to a novel gene, designated 103P2D6 that is over-expressed in multiple cancers listed in Table I.
• Northern blot expression analysis of 103P2D6 gene expression in normal tissues shows a restricted expression pattern in adult tissues.
• Analysis of 103P2D6 expression in normal prostate and prostate tumor xenografts shows over-expression in LAPC-4 and LAPC-9 prostate tumor xenografts.
• the nucleotide (FIG. 2) and amino acid (FIG. 2 and FIG. 3) sequences of 103P2D6 are provided. Portions of the 103P2D6 amino acid sequence show some homologies to ESTs in the dbEST database.
• tissue-related profile of 103P2D6 in normal adult tissues combined with the over-expression observed in prostate and other tumors, shows that 103P2D6 is aberrantly over- expressed in at least some cancers, and thus serves as a useful diagnostic and/or therapeutic target for cancers of the tissues listed in Table I.
• the invention provides polynucleotides corresponding or complementary to all or part of the 103P2D6 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 103P2D6-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 103P2D6 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 103P2D6 genes, mRNAs, or to 103P2D6-encoding polynucleotides.
• Recombinant DNA molecules containing 103P2D6 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 103P2D6 gene products are also provided.
• the invention further provides antibodies that bind to 103P2D6 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker.
• the invention further provides methods for detecting the presence and status of 103P2D6 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 103P2D6.
• a typical embodiment of this invention provides methods for monitoring 103P2D6 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
• the invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 103P2D6 such as prostate cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of 103P2D6 as well as cancer vaccines.
• FIG. 1 shows the 103P2D6 suppression subtractive hybridization (SSH) DNA sequence (SEQ ID NO: 3).
• FIGS. 2A-B shows the nucleotide and amino acid sequences of 103P2D6.
• cDNA and ORF for 103P2D6 clone B, Kozak sequence and start methionine are indicated in bold. See Example 2, infra.
• FIG. 3. shows the amino acid sequence encoded by the open reading frame shown in Fig. 2, and lists the amino acid positions used for proteins/peptides of the invention.
• the 103P2D6 signal sequence is boxed.
• FIG. 4. shows the sequence alignment of 103P2D6 (ORF from clone B) with env protein from human endogenous retroviral HERV-H (FASTA accession: Q9UNM3).
• the 103P2D6 protein sequence has homology to the HERV-H env protein (24.9% identity and 32.8% homology taking account of any gaps).
• FIG. 5A-C shows the northern blot analysis of 103P2D6 expression in various normal human tissues (using the 103P2D6 SSH fragment as a probe) and LAPC xenografts.
• Two multiple tissue northern blots (Clontech) and a xenograft northern blot were probed with the 103P2D6 SSH fragment. Size standards in kilobases (kb) are indicated on the side.
• Each lane contains 2 ⁇ g of mRNA for the normal tissues and 10 ⁇ g of total RNA for the xenograft tissues.
• the results show the expression of 103P2D6 in LAPC xenografts, and not in normal prostate and other tissues. Lanes in FIG.
• Lanes in FIG. 5A represent (1) heart; (2) brain; (3) placenta; (4) lung; (5) liver; (6) skeletal muscle; (7) kidney; (8) pancreas.
• Lanes in FIG. 5B represent (1) spleen; (2) thymus; (3) prostate; (4) testis; (5) ovary; (6) small intestine; (7) colon; (8) leukocytes.
• Lanes in FIG. 5C represent (1) prostate; (2) LAPC-4 AD; (3) LAPC-4 Al; (4) LAPC-9 AD; (5) LAPC-9 Al.
• FIG. 6 shows the northern blot analysis of 103P2D6 expression in various cancer cell lines.
• Lanes represent (1) LAPC-4 AD; (2) LAPC-4 Al; (3) LAPC-9 AD; (4) LAPC-9 Al; (5) LNCaP; (6) PC-3; (7) DU145; (8) TsuPrl; (9) LAPC-4 CL; (10) HT1197; (11) SCaBER; (12) UM-UC-3; (13) TCCSUP; (14) J82; (15) 5637; (16) 293T; (17) RD-ES; (18) PANC-1; (19) BxPC-3; (20) HPAC; (21) Capan-1; (22) SK-CO-1; (23) CaCo-2; (24) LoVo; (25) T84; (26) Colo-205; (27) KCL 22; (28) PFSK- 1; (29) T98G; (30) SK-ES-1; (31) HOS; (32) U2-OS; (33) RD-ES; (34) CALU-1; (35) A427; (3
• TERA-1 TERA-2; (51) A431; (52) HeLa; (53) OV-1063; (54) PA-1; (55) SW626; (56) CAOV-3.
• FIG. 7 shows the northern blot analysis of 103P2D6 expression in various LAPC-4 AD xenografts, including subcutaneously grown xenografts (sc), intratibially grown xenografts (it), and xenografts grown within human bone explants (LAPC-4 AD 2 ) in SOD mice.
• Lanes represent (1) LAPC-4 AD sc; (2) LAPC-4 AD sc; (3) LAPC-4 AD sc; (4) LAPC-4 AD it; (5) LAPC-4 AD it; (6) LAPC-4 AD it; (7) LAPC-4 AD 2 .
• FIG. 8. shows a northern blot analysis of 103P2D6 expression in 9 week old fetal tissues, including 6 organs and whole embryo. Lanes represent (1) brain; (2) heart; (3) kidney; (4) liver; (5) lung; (6) muscle; (7) whole embryo.
• FIG. 9 shows a RT-PCR Expression analysis of 103P2D6. cDNAs generated using pools of tissues from multiple normal and cancer tissues were normalized using beta-actin primers and used to study the expression of 103P2D6. Aliquots of the RT-PCR mix after 26 (upper portion of this figure) and 30 cycles (lower portion of this figure) were run on the agarose gel to allow semi-quantitative evaluation of the levels of expression between samples.
• the first strand cDNAs in the various lanes of this figure are as follows: Lane 1 (VP-1) contains liver, lung, and kidney first strand cDNA from normal tissues; lane 2 (VP-2) stomach, spleen, and pancreas from normal tissues; lane 3 (xenograft tissue pool) LAPC4AD, LAPC4AI, LAPC9AD, and LAPC9AI; lane 4 is normal prostate tissue pool; lane 5 is prostate cancer tissue pool; lane 6 is bladder cancer tissue pool; lane 7 is kidney cancer tissue pool; lane 8 is colon cancer tissue pool; lane 9 is from a lung cancer patient; and lane 10 is water blank.
• FIG. 10. shows the results of RT-PCR analysis of 103P2D6 expression in patient-derived cancers.
• Lane 1 contains a sample from normal prostate; lane 2 from normal kidney; lane 3 from a prostate tumor pool; lane 4 from a kidney tumor pool; lane 5 from a bladder tumor pool; lane 6 from HeLa cells; and for lane 7 water was used.
• FIG. 11A-C shows expression of 103P2D6 in pancreatic, colon, and prostate cancer cell lines.
• cell lysates ⁇ 25 ⁇ g
• cell lysates ⁇ 25 ⁇ g
• an anti-103P2D6 pAb Indicated with an arrow is a strong anti-103P2D6 pAb immunoreactive band of approximately 60 kD present in the pancreatic cancer cell lines HP AC and Bx PC-3, the colon cancer cell line CaCo-2, and a less intense band in LAPC9 prostate cancer cells indicative of endogenous 103P2D6 protein expression.
• Bx PC-3 pancreatic cancer cells were stained with anti-103P2D6 pAb (10 ⁇ g/ml) or control rabbit IgG Ab and subjected to flow cytometric analysis following incubation with anti-rabbit IgG- FITC conjugated secondary Ab.
• Bx PC-3 cells stained with the anti-103P2D6 pAb exhibited a fluorescence shift compared to the cells stained with control rabbit IgG, indicating cell surface expression of 103P2D6.
• FIG. 12A-B shows expression of 103P2D6 protein in 293T cells.
• 293T cells were transiently transfected with either pCDNA 3.1 V5-His 103P2D6 plasmid or with empty control vector and harvested 2 days later. Cells were lysed in SDS-PAGE sample buffer and lysates were separated by SDS-PAGE gel and transferred to nitrocellulose. Western blotting was carried out with an anti-103P2D6 rabbit pAb (2 ⁇ g/ml) raised against a peptide encoding amino acids 163-176 in the 103P2D6 extracellular domain.
• Anti-103P2D6 immunoreactive bands were detected by incubation with anti-rabbit-HRP conjugated secondary Ab and developed using enhanced chemiluminescence and exposure to autoradiographic film. Indicated by arrow is a specific anti- 103P2D6 immunoreactive band of approximately 85 kD in 103P2D6-transfected cells but not in control cells.
• 103P2D6 transfected and vector transfected cells were stained with 10 ⁇ g/ml of anti-103P2D6 pAb and subjected to flow cytometry following incubation with anti-rabbit- FITC conjugated secondary Ab. Shown is a fluorescent shift in 103P2D6-transfected cells compared to the vector transfected cells, indicating cell surface expression of 103P2D6 protein.
• FIG. 13 shows the expression of 103P2D6 as assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots.
• 103P2D6 expression was seen in cancers of the kidney, breast, prostate, uterus, ovary, cervix, colon, stomach and rectum.
• 103P2D6 was also found to be highly expressed in the two human cancer cell lines, the CML line K562 and the colorectal carcinoma SW480.
• the expression detected in normal adjacent tissues isolated from diseased tissues but not in normal tissues, isolated from healthy donors, indicates that these tissues are not fully normal and that 103P2D6 may be expressed in early stage tumors and that it has utility as a diagnostic marker.
• Cancer cell lines are, from left to right, HeLa (cervical carcinoma); Daudi (Burkitt's lymphoma); K562 (CML); HL-60 (PML); G361 (melanoma); A549 (lung carcinoma); MOLT-4 (lymphoblastic leuk.); SW480 (colorectal carcinoma); Raji (Burkitt's lymphoma).
• FIG. 14 shows data where RNA was isolated from prostate tumors (T) and their adjacent normal tissues (N) obtained from the following prostate cancer patients (Pt); patient 1, Gleason score 4+5; patient 2, Gleason score 3+4; and, patient 3, Gleason score 4+3.
• NP normal prostate.
• Northern analysis was performed using lO ⁇ g of total RNA for each sample. Expression of 103P2D6 was seen in all three tumor samples tested and their respective normal tissues.
• FIG. 15 provides data for Northern experiments where RNA was isolated from kidney tumors (T) and their adjacent normal tissues (N) obtained from the following kidney cancer patients: Patient 1- Papillary Type, Stage I, Grade 2/4; Patient 2- Invasive papillary carcinoma, Grade 2/4; Patient 3- Clear cell type Grade 1/3, focally 2/3; Patient 4- Clear cell type, stage III, Grade 2/4; Patient 5- Clear cell type, stage III, Grade 3/4; Patient 6- Clear cell type, stage III, Grade 3/4; Patient 7- Clear cell type, Grade III.
• the Northern analysis was performed using lO ⁇ g of total RNA for each sample. Elevated expression of 103P2D6 was observed in kidney tumors and normal adjacent tissues isolated from kidney cancer patients as compared to normal kidney.
• the terms "advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage Cl - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node, metastasis) system.
• AUA American Urological Association
• stage Cl - C2 disease under the Whitmore-Jewett system
• TNM tumor, node, metastasis
• Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base.
• Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
• “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 103P2D6 (either by remov ig the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 103P2D6.
• the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
• analog refers to a molecule that is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 103P2D6-related protein).
• a 103P2D6-related protein e.g. an analog of the 103P2D6 protein can be specifically bound by an antibody or T cell that specifically binds to 103P2D6.
• Antibody is used in the broadest sense. Therefore an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology.
• Anti-103P2D6 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and or one or more complementarity detei nining regions of these antibodies.
• an "antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-103P2D6 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-103P2D6 antibody compositions with polyepitopic specificity.
• codon optimized sequences refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and or optimization of GC content in addition to codon optimization are referred to herein as an "expression enhanced sequences.”
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
• the term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
• cytotoxic agents include, but are not limited to maytansinoids, ytrium, bismuth ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At 211 , 1 131 , 1 125 ,
• Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
• the term "homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
• hybridize As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1% SDS/100 ⁇ g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1% SDS are above 55 degrees C.
• a polynucleotide is said to be "isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 103P2D6 gene or that encode polypeptides other than 103P2D6 gene product or fragments thereof.
• a skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 103P2D6 polynucleotide.
• a protein is said to be "isolated” when physical, mechanical or chemical methods are employed to remove the 103P2D6 protein from cellular constituents that are normally associated with the protein.
• a skilled artisan can readily employ standard purification methods to obtain an isolated 103P2D6 protein.
• an isolated protein can be prepared by chemical means.
• the term "mammal” as used herein refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
• metastatic prostate cancer and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM+ under the TNM system.
• surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality.
• Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone.
• Prostate cancer bone etastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
• monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
• motif as in biological motif of an 103P2D6-related protein refers to any set of amino acids forming part of the primary sequence of a protein, either contiguous or capable of being aligned to certain positions that are generally invariant, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either Immorally or cellularly.
• a particular function e.g. protein-protein interaction, protein-DNA interaction, etc
• modification e.g. that is phosphorylated, glycosylated or amidated
• localization e.g. secretory sequence, nuclear localization sequence, etc.
• polynucleotide means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”.
• a polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T) (as shown for example in SEQ ID NO: 1) can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
• T thymidine
• U uracil
• polypeptide means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or "protein”.
• a "recombinant" DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
• “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
• “Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (PH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dex
• Modely stringent conditions are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above.
• washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
• moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
• 5 x SSC 150 mM NaCl, 15 mM trisodium citrate
• 50 mM sodium phosphate pH 7.6
• 5 x Denhardt's solution 10% dextran sulfate
• 20 mg/mL denatured sheared salmon sperm DNA followed by washing the filters in 1 x SSC at about 37-50°C.
• the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
• transgenic animal e.g., a mouse or rat
• transgene is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
• transgene is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.
• variant refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 103P2D6 protein shown in FIG. 2 and Fig. 3).
• An analog is an example of a variant protein.
• the 103P2D6-related gene and 103P2D6-related protein includes the 103P2D6 genes and proteins specifically described herein, as well as structurally and/or functionally similar variants or analog of the foregoing.
• 103P2D6 peptide analogs generally share at least about 50%, 60%, 70%, 80%, 90% or more amino acid homology (using BLAST criteria).
• 103P2D6 nucleotide analogs preferably share 50%, 60%, 70%, 80%, 90% or more nucleic acid homology (using BLAST criteria). In some embodiments, however, lower homology is preferred so as to select preferred residues in view of species-specific codon preferences for optimized protein expression and production and/or immunogenicity-modulated peptide epitopes tailored to a particular target population, e.g. HLA type, as is appreciated by those skilled in the art.
• the 103P2D6-related proteins of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art.
• 103P2D6 proteins are collectively referred to as the 103P2D6-related proteins, the proteins of the invention, or 103P2D6.
• the term "103P2D6-related protein” refers to a polypeptide fragment or an 103P2D6 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids.
• 103P2D6 exhibits specific properties that are analogous to those found in a family of molecules whose polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular prostate cancer (see, e.g., both its higlily specific pattern of tissue expression as well as its overexpression in prostate cancers as described for example in Example 3).
• CTL reactive cytotoxic T cells
• HTL reactive helper T cells
• anti-polypeptide antibodies are used in well known diagnostic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular prostate cancer (see, e.g., both its higlily specific pattern of tissue expression as well as its overexpression in prostate cancers as described for example in Example 3).
• PSA the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g
• this disclosure of the 103P2D6 polynucleotides and polypeptides allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
• Typical embodiments of diagnostic methods that utilize the 103P2D6 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays that employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies.
• PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol.
• the 103P2D6 polynucleotides described herein can be utilized in the same way to detect 103P2D6 overexpression or the metastasis of prostate and other cancers expressing this gene.
• PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 103P2D6 polypeptides described herein can be utilized to generate antibodies for use in detecting 103P2D6 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
• metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node)
• assays which examine a biological sample for the presence of cells expressing 103P2D6 polynucleotides and/or polypeptides can be used to provide evidence of metastasis.
• a biological sample from tissue that does not normally contain 103P2D6-expressing cells lymph node
• lymph node a biological sample from tissue that does not normally contain 103P2D6-expressing cells
• 103P2D6-ex ⁇ ressing cells such as the 103P2D6 expression seen in LAPC4 and LAPC9
• 103P2D6 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 103P2D6 or express 103P2D6 at a different level are found to express 103P2D6 or have an increased expression of 103P2D6 (see, e.g., the 103P2D6 expression in kidney, lung and colon cancer cells and in patient samples etc. shown in Figs. 4-10).
• artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 103P2D6) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
• PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA
• 103P2D6 polynucleotide fragments and polynucleotide variants are used in an analogous manner.
• typical PSA polynucleotides used in methods of monitoring PSA are probes or primers that consist of fragments of the PSA cDNA sequence.
• primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction.
• variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov-Dec;l l(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubul et al. eds., 1995)).
• Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g. the 103P2D6 polynucleotide shown in SEQ ID NO: 1) under conditions of high stringency.
• PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA.
• 103P2D6 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner.
• This practice of using polypeptide fragments or polypeptide variants to generate antibodies is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995).
• each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive.
• polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533).
• a polypeptide comprising one of the 103P2D6 biological motifs discussed herein or available in the art.
• Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. the 103P2D6 polypeptide shown in SEQ ID NO: 2).
• the 103P2D6 polynucleotides and polypeptides exhibit specific properties that make them useful in diagnosing cancers of the prostate. Diagnostic assays that measure the presence of 103P2D6 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA.
• these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 103P2D6 polynucleotides and polypeptides (as well as the 103P2D6 polynucleotide probes and anti-103P2D6 antibodies used to identify the presence of these molecules) must be employed to confirm metastases of prostatic origin.
• the 103P2D6 polynucleotides disclosed herein have a number of other specific utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in 2q34, the chromosomal region to which the 103P2D6 gene maps (see Example 7 below).
• the . 103P2D6- related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun 28;80(l-2): 63- 9).
• 103P2D6-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 103P2D6.
• the amino acid or nucleic acid sequence of FIG. 2, or fragments thereof can be used to generate an immune response to the 103P2D6 antigen.
• Antibodies or other molecules that react with 103P2D6 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
• One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 103P2D6 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 103P2D6-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 103P2D6 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 103P2D6 gene, mRNA, or to an 103P2D6 encoding polynucleotide (collectively, "103P2D6 polynucleotides").
• T can also be U in Fig. 2.
• Embodiments of a 103P2D6 polynucleotide include: a 103P2D6 polynucleotide having the sequence shown in Fig. 2, the nucleotide sequence of 103P2D6 as shown in Fig 2, wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Fig. 2; or, at least
• 103P2D6 nucleotides comprise, where T can be U:
• nucleotide as well as any peptide encoded thereby, that starts at any of the following positions or ranges, and ends at a higher position or range: 1, 804, a range of 1-804, 805, a range of 805-976; a range of 805-2493; a range of 977-1036, a range of 1037- 1413; a range of 1414-1815; a range of 1816-2493; a range of 2494-4727; wherein a range as used in this section is understood to specifically disclose all whole unit positions thereof.
• Another embodiment of the invention comprises a polynucleotide that encodes a 103P2D6- related protein whose sequence is encoded by the cDNA contained in the plasmids deposited with American Type Culture Collection as Accession No. PTA-1155 or PTA-1895.
• Another embodiment comprises a polynucleotide that hybridizes under stringent hybridization conditions, to the human 103P2D6 cDNA shown in SEQ ID NO: 1 or to a polynucleotide fragment thereof.
• Typical embodiments of the invention disclosed herein include 103P2D6 polynucleotides that encode specific portions of the 103P2D6 mRNA sequence (and tliose which are complementary to such sequences) such as those that encode the protein and fragments thereof, for example of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
• representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 103P2D6 protein shown in Fig. 2 and Fig.
• polynucleotides encoding about amino acid 40 to about amino acid 50 of the 103P2D6 protein shown in Fig. 2 and Fig. 3 polynucleotides encoding about amino acid 50 to about amino acid 60 of the 103P2D6 protein shown in Fig. 2 and Fig. 3
• polynucleotides encoding about amino acid 70 to about amino acid 80 of the 103P2D6 protein shown in Fig. 2 and Fig. 3 polynucleotides encoding about amino acid 80 to about amino acid 90 of the 103P2D6 protein shown in Fig.
• polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100-532 of the 103P2D6 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
• Polynucleotides encoding relatively long portions of the 103P2D6 protein are also within the scope of the invention. Additional illustrative embodiments of the invention disclosed herein include 103P2D6 polynucleotide fragments encoding one or more of the biological motifs contained within the 103P2D6 protein sequence, including one or more of the motif-bearing subsequences of the 103P2D6 protein set forth in Table XIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 103P2D6 that exhibit homology to a known molecule.
• typical polynucleotide fragments can encode one or more of the 103P2D6 N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
• III.A.1 Uses of 103P2D6 Polynucleotides
• the polynucleotides of the preceding paragraphs have a number of different specific uses.
• the human 103P2D6 gene maps to chromosome 4pl2-pl4 as determined using the GeneBridge4 radiation hybrid panel (see Example 7).
• the 103P2D6 gene maps to chromosome 4pl2- pl4, polynucleotides that encode different regions of the 103P2D6 protein are used to characterize cytogenetic abnormalities on chromosome 4, band pl2-pl4 that have been identified as being associated with various cancers.
• chromosomal abnormalities in 4pl2-pl4 including translocations and deletions have been identified as frequent cytogenetic abnormalities in a number of different cancers (see, e.g., Zimonjic, D.B. et al., 1999, Hepatology 29(4): 1208-14; Wu, X. et al., 1995, Cancer Res. 55(3):557-61; Arribas, R. et al., 1999, Lab. Invest. 79(2): 111-22).
• polynucleotides encoding specific regions of the 103P2D6 protein provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in this region of chromosome 2 that may contribute to the malignant phenotype.
• these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
• 103P2D6 was shown to be highly expressed in prostate and other cancers
• 103P2D6 polynucleotides are used in methods assessing the status of 103P2D6 gene products in normal versus cancerous tissues.
• polynucleotides that encode specific regions of the 103P2D6 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 103P2D6 gene, such as such regions containing one or more motifs.
• Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
• SSCP single-strand conformation polymorphism
• nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 103P2D6.
• antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner.
• PNAs peptide nucleic acids
• non-nucleic acid molecules such as phosphorothioate derivatives
• Antisense technology entails the aclministration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells.
• the term "antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 103P2D6. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988).
• the 103P2D6 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action.
• S-oligos are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom.
• the S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-l,2-benzodithiol-3-one-l,l- dioxide, which is a sulfur transfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).
• Additional 103P2D6 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
• the 103P2D6 antisense oligonucleotides of the present invention typically can be RNA or
• 103P2D6 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 103P2D6 mRNA.
• 103P2D6 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5' codons or last 10 3' codons of 103P2D6.
• the antisense molecules are modified to employ ribozymes in the inhibition of 103P2D6 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
• nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts tliereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof.
• Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
• a detectable marker such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme.
• Such probes and primers are used to detect the presence of a 103P2D6 polynucleotide in a sample and as a means for detecting a cell expressing a 103P2D6 protein.
• probes include polypeptides comprising all or part of the human 103P2D6 cDNA sequences shown in FIG. 2.
• primer pairs capable of specifically amplifying 103P2D6 mRNAs are also described in the Examples.
• a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 103P2D6 mRNA.
• the 103P2D6 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 103P2D6 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 103P2D6 polypeptides; as tools for modulating or inhibiting the expression of the 103P2D6 gene(s) and/or translation of the 103P2D6 transcript(s); and as therapeutic agents.
• i ⁇ .A.4.1 Isolation of 103P2D6-Encoding Nucleic Acid Molecules
• the 103P2D6 cDNA sequences described herein enable the isolation of other polynucleotides encoding 103P2D6 gene product(s), as well as the isolation of polynucleotides encoding 103P2D6 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of the 103P2D6 gene product as well as polynucleotides that encode analogs of 103P2D6-related proteins.
• Various molecular cloning methods that can be employed to isolate full length cDNAs encoding an 103P2D6 gene are well known (See, for example, Sambrook, J.
• 103P2D6 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 103P2D6 DNA probes or primers.
• BACs bacterial artificial chromosome libraries
• YACs yeast artificial chromosome libraries
• the invention also provides recombinant DNA or RNA molecules containing an 103P2D6 polynucleotide, fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al, 1989, supra).
• the invention further provides a host-vector system comprising a recombinant DNA molecule containing a 103P2D6 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell.
• suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell).
• suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl, other transferable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 103P2D6 or a fragment, analog or homolog thereof can be used to generate 103P2D6 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
• 103P2D6 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl.
• the host-vector systems of the invention are useful for the production of a 103P2D6 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 103P2D6 and 103P2D6 mutations or analogs. Recombinant human 103P2D6 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 103P2D6-related nucleotide.
• 293T cells can be transfected with an expression plasmid encoding 103P2D6 or fragment, analog or homolog thereof, the 103P2D6 or related protein is expressed in the 293T cells, and the recombinant 103P2D6 protein is isolated using standard purification methods (e.g., affinity purification using anti-103P2D6 antibodies).
• a 103P2D6 coding sequence is subcloned into the retroviral vector pSR MSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 103P2D6 expressing cell lines.
• codons having a usage frequency of less than about 20% in known sequences of the desired host typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons.
• Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as: http ://www. dna. affrc . go . jp/ ⁇ nakamura/codon.html.
• Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression.
• the GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted haiipin secondary mRNA structures.
• Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol, 9:5073-5080 (1989).
• 103P2D6-related proteins Another aspect of the present invention provides 103P2D6-related proteins.
• 103P2D6 proteins comprise a polypeptide having all or part of the amino acid sequence of human 103P2D6 as shown in FIG. 2.
• embodiments of 103P2D6 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 103P2D6 shown in FIG. 2.
• allelic variants of human 103P2D6 share a high degree of structural identity and homology (e.g., 90% or more homology).
• allelic variants of the 103P2D6 protein contain conservative amino acid substitutions within the 103P2D6 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 103P2D6.
• One class of 103P2D6 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 103P2D6 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift.
• Amino acid abbreviations are provided in Table II.
• Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa.
• Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein.
• glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V).
• Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine.
• Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments (see, e.g. Table III herein; pages 13-15 "Biochemistry" 2 nd ED.
• Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 103P2D6 proteins such as polypeptides having amino acid insertions, deletions and substitutions.
• 103P2D6 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis.
• Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 73:4331 (1986); Zoller et al., Nucl.
• Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction.
• scanning amino acids are relatively small, neutral amino acids.
• amino acids include alanine, glycine, serine, and cysteine.
• Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
• 103P2D6 variants, analogs or homologs have the distinguishing attribute of having at least one epitope that is "cross reactive" with a 103P2D6 protein having the amino acid sequence of SEQ ID NO: 2.
• cross reactive means that an antibody or T cell that specifically binds to an 103P2D6 variant also specifically binds to the 103P2D6 protein having the amino acid sequence of SEQ ID NO: 2.
• a polypeptide ceases to be a variant of the protein shown in SEQ ID NO: 2 when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the 103P2D6 protein.
• Another class of 103P2D6-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.
• Another specific class of 103P2D6 protein variants or analogs comprise one or more of the 103P2D6 biological motifs described herein or presently known in the art.
• analogs of 103P2D6 fragments that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2.
• embodiments of the claimed invention include polypeptides containing less than the 532 amino acid sequence of the 103P2D6 protein shown in FIG. 2.
• representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the 103P2D6 protein shown in Fig. 2 and Fig. 3.
• representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of the 103P2D6 protein shown in Fig. 2 and
• polypeptides consisting of about amino acid 10 to about amino acid 20 of the 103P2D6 protein shown in Fig. 2 and Fig. 3 polypeptides consisting of about amino acid 20 to about amino acid 30 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of the 103P2D6 protein shown in Fig. 2 and Fig.
• polypeptides consisting of about amino acid 60 to about amino acid 70 of the 103P2D6 protein shown in Fig. 2 and Fig. 3 polypeptides consisting of about amino acid 70 to about amino acid 80 of the 103P2D6 protein shown in Fig. 2 and Fig. 3
• polypeptides consisting of about amino acid 80 to about amino acid 90 of the 103P2D6 protein shown in Fig. 2 and Fig. 3 polypeptides consisting of about amino acid 90 to about amino acid 100 of the 103P2D6 protein shown in Fig. 2 and Fig. 3, etc. throughout the entirety of the 103P2D6 sequence.
• polypeptides consisting of 10 amino acid stretches of the amino acid sequence of amino acids 100-532 of the 103P2D6 protein.
• polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of the 103P2D6 protehi shown in Fig. 2 and Fig. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
• 103P2D6-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 103P2D6-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of the 103P2D6 protein (or variants, homologs or analogs thereof). IV.A.1 Motif-bearing Protein Embodiments
• Additional illustrative embodiments of the invention disclosed herein include 103P2D6 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within the 103P2D6 polypeptide sequence set forth in FIG. 2 or FIG 3.
• Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available sites (see, e.g.: http://pfam.wustl.edu/: http://searchlauncher.bcm.tmc.edu/seq-search struc-predict.html http://psort.ims.u-tokyo.ac.ip/: http://www.cbs.dtu.dk/; http://www.ebi.ac.uk/interpro/scan.html; http://www.expasy.ch/tools/scnpsitl.html; EpimatrixTM and EpimerTM, Brown University, http://www.brown.edu
• Motif bearing subsequences of the 103P2D6 protein are set forth and identified in Table XIX.
• Table XX sets forth several frequently occurring motifs based on pfam searches (http://pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location info ⁇ nation is included if the motif is relevant for location.
• Polypeptides comprising one or more of the 103P2D6 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 103P2D6 motifs discussed above are associated with growth dysregulation and because 103P2D6 is overexpressed in certain cancers (See, e.g., Table I).
• Casein kinase II, cAMP and cCMP-dependent protein kinase, and Protein Kinase C are enzymes known to be associated with the development of the malignant phenotype (see e.g.
• Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
• proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth hi Tables V- XVIII.
• CTL epitopes can be determined using specific algorithms to identify peptides within an 103P2D6 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV (A) and Table IV (B); EpimatrixTM and EpimerTM, Brown University, http://www.brown.edu Research TB-
• the epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position.
• polypeptides comprising combinations of the ⁇ different motifs set forth in Table XIX, and or, one or more of the predicted CTL epitopes of Table V through Table XVIII, and or, one or more of the T cell binding motifs known in the art.
• Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides.
• embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located).
• the number of N-terminal and or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
• 103P2D6-related proteins are embodied hi many forms, preferably in isolated form.
• a purified 103P2D6 protein molecule will be substantially free of other proteins or molecules that impair the binding of 103P2D6 to antibody, T cell or other ligand.
• the nature and degree of isolation and purification will depend on the intended use.
• Embodiments of a 103P2D6-related proteins include purified 103P2D6-related proteins and functional, soluble 103P2D6-related proteins.
• a functional, soluble 103P2D6 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
• the invention also provides 103P2D6 proteins comprising biologically active fragments of the 103P2D6 amino acid sequence shown in FIG. 2.
• Such proteins exhibit properties of the 103P2D6 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the 103P2D6 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL.
• 103P2D6-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-103P2D6 antibodies, or T cells or in identifying cellular factors that bind to l03P2D6.
• CTL epitopes can be determined using specific algorithms to identify peptides within an 103P2D6 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV (A) and Table IV (B); EpimatrixTM and EpimerTM, Brown University (http://www.brown.edu Research/TB- HIV_Lab/epirnatrix eprmatrix.html); and BDVIAS, http: ⁇ imas.dcrt.nih.gov/).
• peptide epitopes from 103P2D6 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, Al l, A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, the complete amino acid sequence of the 103P2D6 protein was entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above. The HLA peptide motif search algorithm was developed by Dr.
• the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)).
• Selected results of 103P2D6 predicted binding peptides are shown in Tables V- XVIII herein.
• Tables V-XVIII the top 50 ranking candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score.
• the binding score corresponds to the estimated half-time of dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
• every epitope predicted by the BIMAS site, EpimerTM and EpimatrixTM sites, or specified by the HLA class I or class I motifs available in the art or which become part of the art such as set forth in Table IV (A) and Table IV (B) are to be "applied” to the 103P2D6 protein.
• “applied” means that the 103P2D6 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by tliose of skill in the relevant art.
• Every subsequence of the 103P2D6 of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
• 103P2D6 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 103P2D6 with a C-terminal 6XHis and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN).
• the Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 103P2D6 protehi in transfected cells.
• the secreted HIS-tagged 103P2D6 in the culture media can be purified, e.g., using a nickel column using standard techniques. IV.C.1 Modifications of 103P2D6-related Proteins
• 103P2D6-related proteins such as covalent modifications are included within the scope of this invention.
• One type of covalent modification includes reacting targeted amino acid residues of a 103P2D6 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the 103P2D6.
• Another type of covalent modification of the 103P2D6 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention.
• Another type of covalent modification of 103P2D6 comprises linking the 103P2D6 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
• the 103P2D6-related proteins of the present invention can also be modified to form a chimeric molecule comprising 103P2D6 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly.
• a chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof.
• a protein in accordance with the invention can comprise a fusion of fragments of the 103P2D6 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences respectively of FIG. 2.
• Such a chimeric molecule can comprise multiples of the same subsequence of 103P2D6.
• a chimeric molecule can comprise a fusion of a 103P2D6-related protein with a polyliistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind.
• the epitope tag is generally placed at the amino- or carboxyl- terminus of the 103P2D6.
• the chimeric molecule can comprise a fusion of a 103P2D6-related protein with an immunoglobulin or a particular region of an immunoglobulin.
• an immunoglobulin also referred to as an "immunoadhesin”
• a fusion could be to the Fc region of an IgG molecule.
• the Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 103P2D6 polypeptide in place of at least one variable region within an Ig molecule.
• the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule.
• immunoglobulin fusions see, e.g., U.S. Patent No. 5,428,130 issued June 27, 1995. IV.D.) Uses of 103P2D6-related Proteins
• the proteins of the invention have a number of different specific uses. As 103P2D6 is highly expressed in prostate and other cancers, 103P2D6-related proteins are used in methods that assess the status of 103P2D6 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of the 103P2D6 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs).
• perturbations such as deletions, insertions, point mutations etc.
• Exemplary assays utilize antibodies or T cells targeting 103P2D6-related proteins comprising the amino acid residues of one or more of the biological motifs contained within the 103P2D6 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope.
• 103P2D6-related proteins that contain the amino acid residues of one or more of the biological motifs in the 103P2D6 protein are used to screen for factors that interact with that region of 103P2D6.
• 103P2D6 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 103P2D6 protein), for identifying agents or cellular factors that bind to 103P2D6 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
• domain-specific antibodies e.g., antibodies recognizing an extracellular or intracellular epitope of an 103P2D6 protein
• Proteins encoded by the 103P2D6 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to an 103P2D6 gene product.
• Antibodies raised against an 103P2D6 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 103P2D6 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers.
• 103P2D6-related nucleic acids or proteins are also used in generating HTL or CTL responses.
• 103P2D6 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like.
• Antibodies can be labeled and used as immunological imaging reagents capable of detecting 103P2D6- expressing cells (e.g., in radioscintigraphic imaging methods).
• 103P2D6 proteins are also particularly useful in generating cancer vaccines, as further described herein.
• Another aspect of the invention provides antibodies that bind to 103P2D6-related proteins.
• Preferred antibodies specifically bind to a 103P2D6-related protein and do not bind (or bind weakly) to peptides or proteins that are not 103P2D6-related proteins.
• antibodies bind 103P2D6 can bind 103P2D6-related proteins such as the homologs or analogs thereof.
• 103P2D6 antibodies of the invention are particularly useful in prostate cancer diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 103P2D6 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are tlierapeutically useful in treating cancers in which the expression of 103P2D6 is involved, such as advanced or metastatic prostate cancers.
• the invention also provides various immunological assays useful for the detection and quantification of 103P2D6 and mutant 103P2D6-related proteins.
• Such assays can comprise one or more 103P2D6 antibodies capable of recognizing and binding a 103P2D6-related protein, as appropriate.
• These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme- linked immunofluorescent assays (ELIFA), and the like.
• Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
• immunological imaging methods capable of detecting prostate cancer and other cancers expressing 103P2D6 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 103P2D6 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 103P2D6 expressing cancers such as prostate cancer.
• 103P2D6 antibodies are also used in methods for purifying a 103P2D6-related protein and for isolating 103P2D6 homologues and related molecules.
• a method of purifying a 103P2D6- related protein comprises incubating an 103P2D6 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 103P2D6-related protein under conditions that permit the 103P2D6 antibody to bind to the 103P2D6-related protein; washing the solid matrix to eliminate impurities; and eluting the 103P2D6-related protein from the coupled antibody.
• Other uses of the 103P2D6 antibodies of the invention include generating anti-idiotypic antibodies that mimic the 103P2D6 protein.
• antibodies can be prepared by immunizing a suitable mammalian host using a 103P2D6-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
• fusion proteins of 103P2D6 can also be used, such as a 103P2D6 GST-fusion protein.
• a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies.
• a 103P2D6-related protein is synthesized and used as an immunogen.
• naked DNA immunization techniques known in the art are used (with or without purified 103P2D6-related protein or 103P2D6 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al, 1997, Ann. Rev. Immunol. 15: 617-648).
• the amino acid sequence of 103P2D6 as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 103P2D6 protein for generating antibodies.
• hydrophobicity and hydrophilicity analyses of the 103P2D6 amino acid sequence are used to identify hydrophilic regions in the 103P2D6 structure.
• Regions of the 103P2D6 protein that show hnmunogenic structure, as well as other regions and domains can readily be identified using various other methods known in the art, such as Chou- Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson- Wolf analysis.
• each region identified by any of these programs or methods is within the scope of the present invention.
• Methods for the generation of 103P2D6 antibodies are further illustrated by way of the examples provided herein.
• Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art.
• methods for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH or other carrier protein.
• a carrier such as BSA, KLH or other carrier protein.
• direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, EL, are effective.
• Administration of a 103P2D6 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art.
• liters of antibodies can be taken to determine adequacy of antibody formation.
• 103P2D6 monoclonal antibodies can be produced by various means well known in the art.
• immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybrido a technology of Kohler and Milstein or modifications that immortalize antibody- producing B cells, as is generally known.
• Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 103P2D6-related protein.
• the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
• the antibodies or fragments of the invention can also be produced, by recombinant means.
• Regions that bind specifically to the desired regions of the 103P2D6 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin.
• Humanized or human 103P2D6 antibodies can also be produced, and are preferred for use in therapeutic contexts.
• Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences are well known (see for example, Jones et al, 1986, Nature 321: 522-525; Riechmnan et al, 1988, Nature 332: 323-327; Verhoeyen et al, 1988, Science 239: 1534-1536). See also, Carter et al, 1993, Proc. Natl. Acad. Sci. USA 89; 4285 and Sims et al, 1993, J. Immunol. 151: 2296.
• Fully human 103P2D6 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications hi Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id, pp 65-82).
• Fully human 103P2D6 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and Jakobovits et al, published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 2000; and, 6,114598 issued 5 September 2000). This method avoids the in vitro manipulation requhed with phage display technology and efficiently produces high affinity authentic human antibodies.
• Reactivity of 103P2D6 antibodies with an 103P2D6-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 103P2D6-related proteins, 103P2D6-expressing cells or extracts thereof.
• a 103P2D6 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
• bi-specific antibodies specific for two or more 103P2D6 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al. Cancer Res. 53: 2560-2565). VI.1 103P2D6 TRANSGENIC ANIMALS
• Nucleic acids that encode a 103P2D6-related protein can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents.
• cDNA encoding 103P2D6 can be used to clone genomic DNA that encodes 103P2D6. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 103P2D6.
• Methods for generating transgenic animals, particularly animals such as mice or rats have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 issued 12 April 1988, and 4,870,009 issued 26 September 1989.
• particular cells would be targeted for 103P2D6 transgene incorporation with tissue-specific enhancers.
• Transgenic animals that include a copy of a transgene encoding 103P2D6 can be used to examine the effect of increased expression of DNA that encodes 103P2D6. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
• non-human homologues of 103P2D6 can be used to construct a 103P2D6 "knock out" animal that has a defective or altered gene encodhig 103P2D6 as a result of homologous recombination between the endogenous gene encoding 103P2D6 and altered genomic DNA encoding 103P2D6 introduced into an embryonic cell of the animal.
• cDNA that encodes 103P2D6 can be used to clone genomic DNA encoding 103P2D6 in accordance with established techniques.
• a portion of the genomic DNA encoding 103P2D6 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration.
• another gene such as a gene encoding a selectable marker that can be used to monitor integration.
• several kilobases of unaltered flanking DNA are included in the vector (see, e.g., Thomas and Capecchi, Cell. 51:503 (1987) for a description of homologous recombination vectors).
• the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g.,, Li et al.
• the selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g.,, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152).
• aggregation chimeras see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152).
• a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a "knock out" animal.
• Progeny harboring the homologously recombined DNA in then germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for then ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of the 103P2D6 polypeptide.
• Another aspect of tlie present invention relates to methods for detecting 103P2D6 polynucleotides and 103P2D6-related proteins, as well as methods for identifying a cell that expresses 103P2D6.
• the expression profile of 103P2D6 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 103P2D6 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness.
• the status of 103P2D6 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro- dissected samples), Western blot analysis and tissue array analysis.
• the invention provides assays for the detection of 103P2D6 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like.
• Detectable 103P2D6 polynucleotides include, for example, a 103P2D6 gene or fragment thereof, 103P2D6 mRNA, alternative splice variant 103P2D6 mRNAs, and recombinant DNA or RNA molecules that contain a 103P2D6 polynucleotide.
• a number of methods for amplifying and/or detecting the presence of 103P2D6 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
• a method for detecting an 103P2D6 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using an 103P2D6 polynucleotides as sense and antisense primers to amplify 103P2D6 cDNAs therein; and detecting the presence of the amplified 103P2D6 cDNA.
• the sequence of the amplified 103P2D6 cDNA can be determined.
• a method of detecting a 103P2D6 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 103P2D6 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 103P2D6 gene.
• Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequences provided for the 103P2D6 (FIG. 2) and used for this purpose.
• the invention also provides assays for detecting the presence of an 103P2D6 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like.
• Methods for detecting a 103P2D6-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like.
• a method of detecting the presence of a 103P2D6-related protein in a biological sample comprises first contacting the sample with a 103P2D6 antibody, a 103P2D6-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 103P2D6 antibody; and then detecting the binding of 103P2D6-related protein in the sample.
• an assay for identifying a cell that expresses a 103P2D6 gene comprises detecting the presence of 103P2D6 mRNA in the cell.
• Methods for the detection of particular mRNAs in cells include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 103P2D6 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 103P2D6, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
• an assay for identifying a cell that expresses a 103P2D6 gene comprises detecting the presence of 103P2D6-related protein in the cell or secreted by the cell.
• Various methods for the detection of proteins are well known in the art and are employed for the detection of 103P2D6-related proteins and cells that express 103P2D6-related proteins.
• 103P2D6 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 103P2D6 gene expression.
• 103P2D6 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I.
• Identification of a molecule or biological agent that inhibits 103P2D6 expression or over-expression in cancer cells is of therapeutic value.
• such an agent can be identified by using a screen that quantifies 103P2D6 expression by RT-PCR, nucleic acid hybridization or antibody binding.
• Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al. Lab Invest. 77(5): 437-438 (1997) and Isaacs et al. Cancer Surv. 23: 19-32 (1995)).
• examining a biological sample for evidence of dysregulated cell growth allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse.
• the status of 103P2D6 in a biological sample of interest can be compared, for example, to the status of 103P2D6 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not effected by a pathology).
• a corresponding normal sample e.g. a sample from that individual or alternatively another individual that is not effected by a pathology.
• An alteration in the status of 103P2D6 in the biological sample provides evidence of dysregulated cellular growth.
• a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al, J. Comp. Neurol. 1996 Dec 9;376(2):306-14 and U.S. Patent No. 5,837,501) to compare 103P2D6 status in a sample.
• status in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products.
• skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 103P2D6 expressing cells) as well as the, level, and biological activity of expressed gene products (such as 103P2D6 mRNA polynucleotides and polypeptides).
• an alteration in the status of 103P2D6 comprises a change in the location of 103P2D6 and/or 103P2D6 expressing cells and or an increase in 103P2D6 mRNA and/or protein expression.
• 103P2D6 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis.
• Typical protocols for evaluating the status of the 103P2D6 gene and gene products are found, for example in Ausubul et al. eds, 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).
• the status of 103P2D6 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in the 103P2D6 gene), Northern analysis and or PCR analysis of 103P2D6 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 103P2D6 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 103P2D6 proteins and/or associations of 103P2D6 proteins with polypeptide binding partners).
• genomic Southern analysis to examine, for example perturbations in the 103P2D6 gene
• Northern analysis and or PCR analysis of 103P2D6 mRNA to examine, for example alterations in the polynucleotide sequences or expression levels of 103P2D6 mRNAs
• Detectable 103P2D6 polynucleotides include, for example, a 103P2D6 gene or fragment thereof, 103P2D6 mRNA, alternative splice variants, 103P2D6 mRNAs, and recombinant DNA or RNA molecules containing a 103P2D6 polynucleotide.
• the expression profile of 103P2D6 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample.
• the status of 103P2D6 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness.
• the invention provides methods and assays for determining 103P2D6 status and diagnosing cancers that express 103P2D6, such as cancers of the tissues listed in Table I.
• 103P2D6 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue
• assays that evaluate the levels of 103P2D6 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 103P2D6 dysregulation , and can provide prognostic information useful in defining appropriate therapeutic options.
• the expression status of 103P2D6 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness.
• the expression profile makes it useful as an imaging reagent for metastasized disease.
• an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 103P2D6 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
• the status of 103P2D6 in a biological sample can be examined by a number of well-known procedures in the art.
• the status of 103P2D6 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 103P2D6 expressing cells (e.g. those that express 103P2D6 mRNAs or proteins).
• This examination can provide evidence of dysregulated cellular growth, for example, when 103P2D6- expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 103P2D6 in a biological sample are often associated with dysregulated cellular growth.
• one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node).
• evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al. Prostate 42(4): 315-317 (2000);Su et al, Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al, J Urol 1995 Aug;154(2 Pt l):474-8).
• the invention provides methods for monitoring 103P2D6 gene products by determining the status of 103P2D6 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 103P2D6 gene products in a corresponding normal sample.
• the presence of aberrant 103P2D6 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
• the invention provides assays useful in dete ⁇ nining the presence of cancer in an individual, comprising detecting a significant increase in 103P2D6 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue.
• the presence of 103P2D6 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I.
• the presence of significant 103P2D6 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 103P2D6 mRNA or express it at lower levels.
• 103P2D6 status is determined at the protein level rather than at the nucleic acid level.
• such a method comprises determining the level of 103P2D6 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 103P2D6 expressed in a corresponding normal sample.
• the presence of 103P2D6 protein is evaluated, for example, using immunohistochemical methods.
• 103P2D6 antibodies or binding partners capable of detecting 103P2D6 protein expression are used in a variety of assay formats well known in the art for this purpose.
• These perturbations can include insertions, deletions, substitutions and the like.
• Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al, 1999, J. Cutan. Pathol. 26(8):369-378).
• a mutation in the sequence of 103P2D6 may be indicative of the presence or promotion of a tumor.
• Such assays therefore have diagnostic and predictive value where a mutation in 103P2D6 indicates a potential loss of function or increase in tumor growth.
• methylation status of the 103P2D6 gene hi a biological sample.
• Aberrant demetliylation and/or hypermethylation of CpG islands in gene 5' regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes.
• promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al. Am. J. Pathol. 155(6): 1985-1992 (1999)).
• methylation-sensitive restriction enzymes which cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands.
• MSP methylation specific PCR
• MSP methylation specific PCR
• This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification- using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubul et al. eds, 1995.
• Gene amplification is an additional method for assessing the status of 103P2D6.
• Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
• antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
• Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 103P2D6 expression.
• the presence of RT-PCR amplifiable 103P2D6 mRNA provides an indication of the presence of cancer.
• RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use hi the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al, 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al, 1995, Clin. Chem. 41:1687-1688).
• a further aspect of the invention is an assessment of the susceptibiUty that an individual has for developing cancer.
• a method for predicting susceptibiUty to cancer comprises detecting 103P2D6 mRNA or 103P2D6 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 103P2D6 mRNA expression correlates to the degree of susceptibility.
• the presence of 103P2D6 in prostate or other tissue is examined, with the presence of 103P2D6 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor).
• 103P2D6 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations hi the structure of these molecules such as insertions, deletions, substitutions and the like.
• the presence of one or more perturbations in 103P2D6 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
• the invention also comprises methods for gauging tumor aggressiveness.
• a method for gauging aggressiveness of a tumor comprises determining the level of 103P2D6 mRNA or 103P2D6 protein expressed by tumor cells, comparing the level so determined to the level of 103P2D6 mRNA or 103P2D6 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 103P2D6 mRNA or 103P2D6 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness.
• aggressiveness of a tumor is evaluated by determining the extent to which 103P2D6 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors.
• Another embodiment is the evaluation of the integrity of 103P2D6 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
• methods for observing the progression of a malignancy in an individual over time comprise determining the level of 103P2D6 mRNA or 103P2D6 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 103P2D6 mRNA or 103P2D6 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 103P2D6 mRNA or 103P2D6 protein expression in the tumor sample over time provides information on the progression of the cancer.
• the progression of a cancer is evaluated by dete ⁇ nining 103P2D6 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer.
• the above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art.
• another embodiment of the invention is directed to methods for observing a coincidence between the expression of 103P2D6 gene and 103P2D6 gene products (or perturbations in 103P2D6 gene and 103P2D6 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample.
• factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Booking et al, 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein, 1995, Hum. Pathol.
• methods for observing a coincidence between the expression of 103P2D6 gene and 103P2D6 gene products (or perturbations in 103P2D6 gene and 103P2D6 gene products) and another factor associated with malignancy entails detecting the overexpression of 103P2D6 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 103P2D6 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression).
• the expression of 103P2D6 and PSA mRNA in prostate tissue is examined, where the coincidence of 103P2D6 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibiUty or the emergence or status of a prostate tumor.
• Standard methods for the detection and quantification of 103P2D6 mRNA include in situ hybridization using labeled 103P2D6 riboprobes, Northern blot and related techniques using 103P2D6 polynucleotide probes, RT-PCR analysis using primers specific for 103P2D6, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like.
• semi-quantitative RT-PCR is used to detect and quantify 103P2D6 mRNA expression.
• primers capable of amptifying 103P2D6 can be used for this purpose, including but not limited to the various primer sets specifically described herein.
• polyclonal or monoclonal antibodies specifically reactive with the wild-type 103P2D6 protein can be used in an immunohistochemical assay of biopsied tissue.
• the 103P2D6 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 103P2D6, as well as pathways activated by 103P2D6 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the "two-hybrid assay"). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed.
• interaction trap systems also referred to as the "two-hybrid assay”
• peptide libraries can be screen peptide libraries to identify molecules that interact with 103P2D6 protein sequences.
• peptides that bind to a molecule such as 103P2D6 are identified by screening libraries that encode a random or controlled collection of amino acids.
• Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the protein of interest.
• peptides having a wide variety of uses are thus identified without any prior information on the structure of the expected ligand or receptor molecule.
• Typical peptide libraries and screening methods that can be used to identify molecules that interact with 103P2D6 protein sequences are disclosed for example in U.S.
• cell lines that express 103P2D6 are used to identify protein-protein interactions mediated by 103P2D6. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).
• 103P2D6 protein can be hnmunoprecipitated from 103P2D6-expressing cell lines using anti-103P2D6 antibodies.
• antibodies against His-tag can be used in a cell line engineered to express 103P2D6 (vectors mentioned above).
• the hnmunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35 S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
• Small molecules and ligands that interact with 103P2D6 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 103P2D6's ability to mediate phosphorylation and de-phosphorylation, second messenger signaling or tumorigenesis. Similarly, ligands that regulate 103P2D6 function can be identified based on then ability to bhid 103P2D6 and activate a reporter construct. Typical methods are discussed for example hi U.S. Patent No. 5,928,868 issued 27 July 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule.
• cells engineered to express a fusion protein of 103P2D6 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein.
• the cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins.
• Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying both activators and inhibitors of 103P2D6.
• An embodiment of this invention comprises a method of screening for a molecule that interacts with an 103P2D6 amino acid sequence shown in FIG. 2 and FIG. 3, comprising the steps of contacting a population of molecules with the 103P2D6 amino acid sequence, allowing the population of molecules and the 103P2D6 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 103P2D6 amino acid sequence, and then separating molecules that do not interact with the 103P2D6 amino acid sequence from molecules that do.
• the method further comprises purifying a molecule that interacts with the 103P2D6 amino acid sequence.
• the identified molecule can be used to modulate a function performed by 103P2D6.
• the 103P2D6 amino acid sequence is contacted with a library of peptides.
• 103P2D6 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As discussed herein, it is possible that 103P2D6 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
• therapeutic approaches that inhibit the activity of the 103P2D6 protein are useful for patients suffering a cancer that expresses 103P2D6.
• These therapeutic approaches generally fall into two classes.
• One class comprises various methods for inhibiting the binding or association of the 103P2D6 protein with its binding partner or with others proteins.
• Another class comprises a variety of methods for inhibiting the transcription of the 103P2D6 gene or translation of 103P2D6 mRNA.
• 103P2D6 is an attractive target for antibody-based therapeutic strategies.
• a number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies).
• 103P2D6 is expressed by cancer cells of various lineages and not by corresponding normal cells, systemic administration of 103P2D6-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues.
• Antibodies specifically reactive with domains of 103P2D6 are useful to treat 103P2D6-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
• 103P2D6 antibodies can be introduced into a patient such that the antibody binds to 103P2D6 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells.
• Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 103P2D6, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
• antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of the 103P2D6 sequence shown in FIG. 2.
• cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 103P2D6), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
• compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art.
• typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-103P2D6 antibody) that binds to a marker (e.g. 103P2D6) expressed, accessible to binding or localized on the cell surfaces.
• a targeting agent e.g. an anti-103P2D6 antibody
• a marker e.g. 103P2D6
• a typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 103P2D6, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 103P2D6 epitope, and, exposing the cell to the antibody-agent conjugate.
• Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
• Cancer immunotherapy using anti-103P2D6 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, includmg but not limited to colon cancer (Arlen et al, 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al, 1997, Blood 90:3179-3186, Tsunenari et al, 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al, 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al, 1996, J. Immunother. Emphasis Tumor Immunol.
• Some therapeutic approaches involve conjugation of naked antibody to a toxin, such as the conjugation of 131 I to anti-CD20 antibodies (e.g., RituxanTM, IDEC Pharmaceuticals Corp.), while others involve co- administration of antibodies and other therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel (Genentech, Inc.).
• a toxin such as the conjugation of 131 I to anti-CD20 antibodies (e.g., RituxanTM, IDEC Pharmaceuticals Corp.)
• HerceptinTM tacuzumab
• paclitaxel Genetech, Inc.
• 103P2D6 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation.
• antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
• Cancer patients can be evaluated for the presence and level of 103P2D6 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 103P2D6 imaging, or other techniques that reliably indicate the presence and degree of 103P2D6 expression.
• Anti-103P2D6 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic.
• anti-103P2D6 monoclonal antibodies mAbs
• ADCC antibody-dependent cell cytotoxicity
• anti-103P2D6 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 103P2D6.
• Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis.
• the mechanism(s) by which a particular anti-103P2D6 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
• preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bhid specifically to the target 103P2D6 antigen with high affinity but exhibit low or no antigenicity in the patient.
• Therapeutic methods of the invention contemplate the administration of single anti-103P2D6 mAbs as well as combinations, or cocktails, of different mAbs.
• Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects.
• anti-103P2D6 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g, IL-2, GM-CSF), surgery or radiation.
• the anti-103P2D6 mAbs are administered in then "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
• Anti-103P2D6 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell.
• Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like.
• Treatment generally involves repeated administration of the anti-103P2D6 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated.
• IV intravenous injection
• an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti- 103P2D6 mAb preparation represents an acceptable dosing regimen.
• the initial loading dose is administered as a 90 minute or longer infusion.
• the periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated.
• various factors can influence the ideal dose regimen in a particular case.
• Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 103P2D6 expression in the patient, the extent of circulating shed 103P2D6 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
• patients should be evaluated for the levels of 103P2D6 in a given sample (e.g. the levels of circulating 103P2D6 antigen and or 103P2D6 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc.
• levels of 103P2D6 in a given sample e.g. the levels of circulating 103P2D6 antigen and or 103P2D6 expressing cells
• Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (such as serum PSA levels in prostate cancer therapy).
• the invention further provides cancer vaccines comprising a 103P2D6-related protehi or 103P2D6-related nucleic acid.
• cancer vaccines prevent and/or treat 103P2D6-expressing cancers without creating non-specific effects on non-target tissues.
• the use of a tumor antigen in a vaccine that generates humoral and or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al, 1995, Int. J. Cancer 63:231-237; Fong et al, 1997, J. Immunol. 159:3113-3117).
• Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses dhected against cancer cells expressing 103P2D6.
• Constructs comprising DNA encoding a 103P2D6-related protein immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 103P2D6 protein/immunogen.
• a vaccine comprises a 103P2D6-related protein. Expression of the 103P2D6-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear 103P2D6 protein.
• Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address www, genweb . com) .
• Such methods can be readily practiced by employing a 103P2D6-related protein, or an 103P2D6-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 103P2D6 immunogen (which typically comprises a number of antibody or T cell epitopes).
• Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g, Heryln et al, Ann Med 1999 Feb;31(l):66-78; Maruyama et al. Cancer Immunol Immunother 2000 Jun;49(3): 123-32) Briefly, such methods of generating an immune response (e.g.
• humoral and/or cell-mediated in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in the 103P2D6 protein shown in SEQ ID NO: 2 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope).
• an immunoreactive epitope e.g. an epitope present in the 103P2D6 protein shown in SEQ ID NO: 2 or analog or homolog thereof
• the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope).
• the 103P2D6 immunogen contains a biological motif.
• CTL epitopes can be determined using specific algorithms to identify peptides within 103P2D6 protein that are capable of optimally binding to specified HLA alleles (e.g.
• the 103P2D6 immunogen contains one or more amino acid sequences identified using one of the pertinent analytical techniques well known in the art, such as the sequences shown in Tables V- XVIII or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif (e.g.
• HLA Class I binding grove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long.
• HLA Class II binding groove is essentially open ended; therefore a of about 9 or more amino acids can be bound by an HLA Class II molecule.
• HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide.
• the amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl te ⁇ nini of a motif- bearing sequence.
• HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24, or 25 amino acids long, but can be longer than 25 amino acids.
• Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. the 103P2D6 protein) so that an immune response is generated.
• a protein e.g. the 103P2D6 protein
• a typical embodiment consists of a method for generating an immune response to 103P2D6 in a host, by contacting the host with a sufficient amount of at least one 103P2D6 B cell or cytotoxic T-cell epitope or analog tliereof; and at least one periodic interval thereafter re-contacting the host with the 103P2D6 B cell or cytotoxic T-cell epitope or analog thereof.
• a specific embodiment consists of a method of generating an immune response against a 103P2D6-related protein or a man-made multiepitopic peptide comprising: administering 103P2D6 immunogen (e.g.
• Such vaccine preparations further contain a suitable adjuvant (see, e.g, U.S. Patent No. 6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; see, e.g, Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al. Immunity 1994 1(9): 751-761 and Alexander et al, Immunol. Res. 1998 18(2): 79-92).
• a suitable adjuvant see, e.g, U.S. Patent No. 6,146,635
• a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; see, e.g, Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al. Immunity 1994 1(9): 751-761 and Alexander et
• An alternative method comprises generating an immune response in an individual against a 103P2D6 immunogen by: aciministering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes an 103P2D6 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g, U.S. Patent No. 5,962,428).
• the DNA can be dissociated from an infectious agent.
• a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered.
• viral gene delivery systems are used to deliver a 103P2D6-related nucleic acid molecule.
• Various viral gene delivery systems that can be used hi the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8:658-663).
• Non-viral delivery systems can also be employed by introducing naked DNA encoding a 103P2D6-related protein into the patient (e.g, intramuscularly or intradermally) to induce an anti-tumor response.
• the full- length human 103P2D6 cDNA is employed.
• 103P2D6 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
• APCs antigen presenting cells
• dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL- 12, and are thus highly specialized antigen presenting cells.
• PSMA prostate-specific membrane antigen
• dendritic cells can be used to present 103P2D6 peptides to T cells in the context of MHC class I or II molecules.
• autologous dendritic cells are pulsed with 103P2D6 peptides capable of binding to MHC class I and/or class II molecules.
• dendritic cells are pulsed with the complete 103P2D6 protein.
• Yet another embodiment involves engineering the overexpression of the 103P2D6 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al, 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al, 1996, Cancer Res.
• 103P2D6 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
• immune modulators such as GM-CSF
• Anti-idiotypic anti-103P2D6 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 103P2D6-related protein.
• anti-idiotypic antibodies are well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-103P2D6 antibodies that mimic an epitope on a 103P2D6-related protein (see, for example, Wagner et al, 1997, Hybridoma 16: 33-40; Foon et al, 1995, J. Clin. Invest. 96:334- 342; Herlyn et al, 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
• the invention includes various methods and compositions for inhibiting the binding of 103P2D6 to its binding partner or its association with other ⁇ rotein(s) as well as methods for inhibiting 103P2D6 function.
• a recombinant vector that encodes single chahi antibodies that specifically bind to 103P2D6 are introduced into 103P2D6 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-103P2D6 antibody is expressed intracellularly, binds to 103P2D6 protein, and thereby inhibits its function.
• Methods for engineering such intracellular single chain antibodies are well known.
• intracellular antibodies also known as "intrabodies” are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).
• Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g, Richardson et al, 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al, 1994, J. Biol. Chem. 289: 23931- 23936; Deshane et al, 1994, Gene Ther. 1: 332-337).
• Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide.
• single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region.
• Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment.
• intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif.
• Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal.
• Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane.
• Intrabodies can also be targeted to exert function in the cytosol.
• cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
• intrabodies are used to capture 103P2D6 in the nucleus, thereby preventing its activity within the nucleus.
• Nuclear targeting signals are engineered into such 103P2D6 intrabodies in order to achieve the desired targeting.
• Such 103P2D6 intrabodies are designed to bind specifically to a particular 103P2D6 domain.
• cytosolic intrabodies that specifically bind to the 103P2D6 protein are used to prevent 103P2D6 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g, preventing 103P2D6 from forming transcription complexes with other factors).
• the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer.
• an appropriate tumor-specific promoter and/or enhancer In order to target intrabody expression specifically to prostate, for example, the PSA promoter and or promoter/enhancer can be utilized (See, for example, U.S. Patent No. 5,919,652 issued 6 July 1999).
• recombinant molecules bind to 103P2D6 and thereby inhibit 103P2D6 function.
• these recombinant molecules prevent or inhibit 103P2D6 from accessing/binding to its binding partner(s) or associating with other protein(s).
• Such recombinant molecules can, for example, contain the reactive part(s) of a 103P2D6 specific antibody molecule.
• the 103P2D6 binding domain of a 103P2D6 binding partner is engineered into a dimeric fusion protehi, whereby the fusion protein comprises two 103P2D6 ligand binding domains linked to the Fc portion of a human IgG, such as human IgGl.
• Such IgG portion can contain, for example, the C H 2 and C H 3 domains and the hinge region, but not the C H 1 domain.
• Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 103P2D6, whereby the dimeric fusion protein specifically binds to 103P2D6 and blocks 103P2D6 interaction with a binding partner.
• Such dimeric fusion proteins are further combined into multimeric proteins using known antibody Unking technologies.
• the present invention also comprises various methods and compositions for inhibiting the transcription of the 103P2D6 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 103P2D6 mRNA into protein.
• a method of inhibiting the transcription of the 103P2D6 gene comprises contacting the 103P2D6 gene with a 103P2D6 antisense polynucleotide.
• a method of inhibiting 103P2D6 mRNA translation comprises contacting the 103P2D6 mRNA with an antisense polynucleotide.
• a 103P2D6 specific ribozyme is used to cleave the 103P2D6 message, thereby inhibiting translation.
• antisense and ribozyme based methods can also be directed to the regulatory regions of the 103P2D6 gene, such as the 103P2D6 promoter and/or enhancer elements.
• proteins capable of inhibiting a 103P2D6 gene transcription factor are used to inhibit 103P2D6 mRNA transcription.
• the various polynucleotides and compositions useful in the aforementioned methods have been described above.
• the use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
• Other factors that inhibit the transcription of 103P2D6 by interfering with 103P2D6 transcriptional activation are also useful to treat cancers expressing 103P2D6.
• factors that interfere with 103P2D6 processing are useful to treat cancers that express 103P2D6. Cancer treatment methods utilizing such factors are also within the scope of the invention.
• Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 103P2D6 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 103P2D6 inhibitory molecules).
• 103P2D6 i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 103P2D6 inhibitory molecules.
• a number of gene therapy approaches are known in the art.
• Recombinant vectors encoding 103P2D6 antisense polynucleotides, ribozymes, factors capable of interfering with 103P2D6 transcription, and so forth, can be delivered to target tumor ceUs using such gene therapy approaches.
• the above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens.
• the therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for aU patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.
• the anti-tumor activity of a particular composition can be evaluated using various in vitro and in vivo assay systems, fn vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 103P2D6 to a binding partner, etc.
• a 103P2D6 therapeutic composition can be evaluated in a suitable animal model.
• xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SOD mice (Klein et al, 1997, Nature Medicine 3: 402-408).
• PCT Patent AppUcation W098/16628, Sawyers et al, pubUshed April 23, 1998 describes various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease.
• Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like. In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions.
• xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
• Suitable carriers include any material that when combined with the therapeutic composition retains the anti- tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16 th Edition, A. Osal, Ed, 1980).
• Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site.
• Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, inttaperitoneal, intramuscular, infratumor, intradermal, intraorgan, orthotopic, and the like.
• a preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP.
• Therapeutic protein preparations can be lyopliilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
• kits are also within the scope of the invention.
• Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the metliod.
• the container(s) can comprise a probe that is or can be detectably labeled.
• probe can be an antibody or polynucleotide specific for a 103P2D6-related protein or a 103P2D6 gene or message, respectively.
• the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.
• a reporter-means such as a biotin-binding protein, such as avidin or streptavidin
• the kit can include all or part of the amino acid sequence of FIG. 2 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.
• the kit of the invention will typically comprise the container described above and one or more other containers comprising materials deshable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
• a label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.
• pl03P2D6-B (clone B) has been deposited under the requirements of the Budapest Treaty on May 19, 2000 with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209 USA, and has been identified as ATCC Accession No. PTA-1895.
• pl03P2D6-2 (clone 2) has been deposited under the requhements of the Budapest Treaty on January 6, 2000 with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209 USA, and has been identified as ATCC Accession No. PTA-1155.
• Example 1 SSH-generated Isolation of a cDNA Fragment of the 103P2D6 Gene
• mice that harbored LAPC-4 AD xenografts were castrated when the tumors reached a size of 1 cm in diameter. The tumors regressed in size and temporarily stopped producing the androgen dependent protein PSA. Seven to fourteen days post-castration, PSA levels were detectable again in the blood of the mice. Eventually such tumors develop an Al phenotype and start growing again in the castrated males. Tumors were harvested at different time points after castration to identify genes that are turned on or off during the transition to androgen independence.
• SSH Suppression subtractive hybridization
• 103P2D6 encodes a transmembrane protein that exhibits tumor-specific expression. Expression analysis of 103P2D6 indicates that it is exclusively expressed in cancer of the prostate and other cancer tissues. 103P2D6 is expressed in fetal heart, kidney and lung, but not in normal adult tissues. The expression of 103P2D6 in cancer provides evidence that this protein has a functional role in tumor progression and/or initiation. It is possible that 103P2D6 functions as a receptor involved in activating or modulating proliferation signals involved in tumorigenesis and regulation of cell growth. As is further described herein, the 103P2D6 gene and protein have been characterized using a number of analytical approaches.
• analyses of nucleotide coding and amino acid sequences were conducted in order to identify potentially related molecules, as well as recognizable structural domains, topological features, and other elements within the 103P2D6 mRNA and protein structures.
• Northern blot analyses of 103P2D6 mRNA expression were conducted in order to establish the range of normal and cancerous tissues expressing 103P2D6 message.
• LAPC Xenografts and Human Tissues were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craft et al, 1999, Cancer Res. 59: 5030-5036). Androgen dependent and independent LAPC-4 xenografts (LAPC-4 AD and Al, respectively) and LAPC-9 xenografts (LAPC-9 AD and Al, respectively) were grown in intact male SOD mice or in castrated males, respectively, and were passaged as small tissue chunks in recipient males.
• LAPC-4 Al xenografts were derived from LAPC-4 AD tumors and LAPC-9 Al xenografts were derived from LAPC-9 AD tumors.
• male mice bearing LAPC AD tumors were castrated and maintained for 2-3 months. After the LAPC tumors re-grew, the tumors were harvested and passaged in castrated males or in female SOD mice.
• Human cell lines e.g, HeLa
• DMEM fetal calf serum
• Tumor tissue and cell lines were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/ g tissue or 10 ml/ 10 s cells to isolate total RNA.
• Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
• Adaptor 1 5 'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3 ' (SEQ ID NO: 8)
• 3OGCCCGTCCTAG5' (SEQ ID NO: 9)
• Adaptor 2 5OTAATACGACTCACTATAGGGCAGCGTGGTCGCGCGGCCGAG3' (SEQ ID NO:10)
• 3'CGGCTCCTAG5' (SEQ ID NO: 11)
• PCR primer 1 5 'CTAATACGACTCACTATAGGGC3 ' (SEQ ID NO: 12)
• Nested primer CNP 1 :
• SSH Suppression subtractive hybridization
• Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 ⁇ g of poly(A) + RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs. at 37°C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
• Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant xenograft source (see above) with a mix of digested cDNAs derived from human benign prostatic hyperplasia (BPH), the human cell lines HeLa, 293, A431, Colo205, and mouse liver.
• BPH human benign prostatic hyperplasia
• Tester cDNA was generated by diluting 1 ⁇ l of Dpn II digested cDNA from the relevant xenograft source (see above) (400 ng) in 5 ⁇ l of water. The diluted cDNA (2 ⁇ l, 160 ng) was then ligated to 2 ⁇ l of Adaptor 1 and Adaptor 2 (10 ⁇ M), in separate ligation reactions, in a total volume of 10 ⁇ l at 16°C overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 ⁇ l of 0.2 M EDTA and heating at 72°C for 5 min.
• the first hybridization was performed by adding 1.5 ⁇ l (600 ng) of driver cDNA to each of two tubes containing 1.5 ⁇ l (20 ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final volume of 4 ⁇ l, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98°C for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68°C. The two hybridizations were then mixed together with an additional 1 ⁇ l of fresh denatured driver cDNA and were allowed to hybridize overnight at 68°C. The second hybridization was then diluted in 200 ⁇ l of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70°C for 7 min. and stored at -20°C.
• PCR 1 was conducted using the following conditions: 75°C for 5 min, 94°C for 25 sec, then 27 cycles of 94°C for 10 sec, 66°C for 30 sec, 72°C for 1.5 min. Five separate primary PCR reactions were performed for each experiment.
• PCR 2 was performed using 10-12 cycles of 94°C for 10 sec, 68°C for 30 sec, and 72°C for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
• PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampiciUin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
• Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NO-CGAP databases.
• SSH clones Two SSH experiments described in the Materials and Methods, supra, led to the isolation of numerous candidate gene fragment clones (SSH clones). All candidate clones were sequenced and subjected to homology analysis against all sequences in the major pubUc gene and EST databases in order to provide information on the identity of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression. In general, gene fragments that had no homology to any known sequence in any of the searched databases, and thus considered to represent novel genes, as well as gene fragments showing homology to previously sequenced expressed sequence tags (ESTs), were subjected to differential expression analysis by RT-PCR and/or northern analysis.
• ESTs previously sequenced expressed sequence tags
• a partial 103P2D6 cDNA clone (clone 2) of 1687 base pahs (bp) was cloned from an LAPC-4 AD cDNA library (Lambda ZAP Express, Stratagene).
• a full-length 103P2D6 cDNA clone (Fig. 2) (clone B) of 4728 base pairs (b.p.) was cloned from a human fetal brain library (Pangene Inc.).
• the cDNA encodes a putative open reading frame (ORF) of 563 amino acids. Its calculated molecular weight (MW) is 63.4 kDa and its pi is 8.15.
• 103P2D6 shows 24.9% identity and 32.8% homology, taking account of any gaps, to an Envelope protein (Q9UNM3) isolated from a human endogenous retroviral protein HERV-H (Virology 1999, 258:441).
• Q9UNM3 Envelope protein
• HERV-H Human endogenous retroviral protein
• ATCC Manassas, VA
• plasmid pl03P2D6-B plasmid pl03P2D6-B
• accession No. PTA-1895 The partial 103P2D6 cDNA (clone 2) was deposited on January 6, 2000, with the American Type Culture Collection (ATCC; Manassas, VA) as plasmid pl03P2D6-2, and has been assigned Accession No. PTA-1155.
• 103P2D6 mRNA expression in normal human tissues was analyzed by northern blotting of multiple tissue blots (Clontech; Palo Alto, California), comprising a total of 16 different normal human tissues, using labeled 103P2D6 SSH fragment (Example 1) as a probe.
• RNA samples were quantitatively normalized with a ⁇ -actin probe.
• LAPC-4 xenografts More detailed analysis of the LAPC-4 xenografts shows that 103P2D6 is expressed at equal levels whether the xenografts are grown subcutaneously (LAPC-4 sc) or within the tibia of mice (LAPC-4 AD it) (Fig. 7). Expression was also detected in a xenograft that was grown within human bone explants in SOD mice (the LAPC-4 AD 2 ). This indicates that bone growth of these prostate cancer tissues does not diminish then expression. Expression of 103P2D6 was detected in several cancer cell lines derived from prostate
• 103P2D6 Expression of 103P2D6 was assayed in a panel of human cancers (T) and then respective matched normal tissues (N) on RNA dot blots (Fig. 13). 103P2D6 expression was seen in cancers of the kidney, breast, prostate, uterus, ovary, cervix, colon, stomach and rectum. 103P2D6 was also found to be highly expressed in the two human cancer cell lines, the CML line K562 and the colorectal carcinoma SW480. The expression detected in normal adjacent tissues (isolated from diseased tissues) but not in normal tissues, isolated from healthy donors, indicates that these tissues are not fully normal and that 103P2D6 may be expressed in early stage tumors and that it has utility as a diagnostic marker. FIG.
• RNA was isolated from prostate tumors (T) and their adjacent normal tissues (N) obtained from the following prostate cancer patients (Pt); patient 1, Gleason score 4+5; patient 2, Gleason score 3+4; and, patient 3, Gleason score 4+3.
• Northern analysis was performed using lO ⁇ g of total RNA for each sample. Expression of 103P2D6 was seen in all three tumor samples tested and then respective normal tissues. Data from a Northern analysis where RNA was isolated from kidney tumors (T) and their adjacent normal tissues (N) obtained from kidney cancer patients is shown in Fig. 15.
• the patient specifications are as follows: Patient 1- Papillary Type, Stage I, Grade 2/4; Patient 2- Invasive papillary carcinoma, Grade 2/4; Patient 3- Clear cell type Grade 1/3, focally 2/3; Patient 4- Clear cell type, stage III, Grade 2/4; Patient 5- Clear cell type, stage III, Grade 3/4; Patient 6- Clear cell type, stage III, Grade 3/4; Patient 7- Clear cell type, Grade III.
• the Northern analysis was performed using lO ⁇ g of total RNA for each sample. Elevated expression of 103P2D6 was observed in kidney tumors and normal adjacent tissues isolated from kidney cancer patients as compared to normal kidney.
• RNA was isolated from bladder cancers and adjacent normal tissue obtained from the bladder cancer patients.
• the Northern analysis was performed using lO ⁇ g of total RNA for each sample. Expression of 103P2D6 was seen in bladder tumor but not in normal adjacent tissue.
• First strand cDNAs can be generated from 1 ⁇ g of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used, which included an incubation for 50 min at 42°C with reverse transcriptase, followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume can be increased to 200 ⁇ l with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
• First strand cDNA (5 ⁇ l) were amplified in a total volume of 50 ⁇ l containing 0.4 ⁇ M primers, 0.2 ⁇ M each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl 2 , 50 mM KC1, pH8.3) and IX Klentaq DNA polymerase (Clontech). Five ⁇ l of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis.
• PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94°C for 15 sec, followed by a 18, 20, and 22 cycles of 94°C for 15, 65°C for 2 min, 72°C for 5 sec. A final extension at 72°C was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. ⁇ -actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal ⁇ -actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
• RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were subsequently normalized using beta-actin PCR. The highest expression was observed in colon cancer pool, xenograft pool, and a lung cancer patient. Lower levels of expression were also observed normal prostate, prostate cancer, bladder cancer, and kidney cancer tissue pools.
• RT-PCR was used to analyze expression of 103P2D6 in normal tissues and in patient-derived cancers.
• First strand cDNAs were generated from 1 ⁇ g of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturers protocol was used and included an incubation for 50 min at 42°C with reverse transcriptase followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume was increased to 200 ⁇ l with water prior to normalization.
• First strand cDNAs were prepared from normal prostate, normal kidney and HeLa cancer cells, as well as a prostate tumor pool, a kidney tumor pool and a bladder tumor pool. The tumor pools were prepared from patient-derived tumor tissue. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR was performed using primers to 103P2D6.
• the 103P2D6 primers used for RT-PCR were:
• Protein Expression Analysis Expression of 103P2D6 protein was analyzed in pancreatic, colon, and prostate cancer cell lines using both western blot and flow cytometric analysis. As shown in FIG. 11A-B, cell lysates ( ⁇ 25 ⁇ g) from the indicated cell lines were separated by SDS-PAGE and subjected to Western blot analysis using an anti-103P2D6 pAb (see Example 4, below). Indicated with an arrow is a strong anti-103P2D6 pAb immunoreactive band of approximately 60 kD present in the pancreatic cancer cell lines HP AC and Bx PC-3, the colon cancer cell line CaCo-2, and a less intense band in LAPC9 prostate cancer cells, indicative of endogenous 103P2D6 protein expression. Also indicated with an arrow is the 85 kD immunoreactive band present in 293T cells transfected with V5-His tagged 103P2D6 cDNA.
• Bx PC-3 pancreatic cancer cells were stained with anti-103P2D6 pAb (10 ⁇ g/ml) or control rabbit IgG Ab and subjected to flow cytometric analysis following incubation with anti-rabbit IgG- FITC conjugated secondary Ab.
• Bx PC-3 cells stained with the anti-103P2D6 pAb exhibited a fluorescence shift compared to the cells stained with control rabbit IgG, indicating cell surface expression of 103P2D6. The results are shown in FIG. 1 IC.
• Example 4 Generation of 103P2D6 Polyclonal Antibodies
• a peptide was synthesized corresponding to amino acids 163-176 (DVTNESRNDDDDTS) of the 103P2D6 protein sequence.
• the peptide was coupled to Keyhole limpet hemacyanin (KLH) and used to immunize a rabbit as follows.
• KLH Keyhole limpet hemacyanin
• the rabbit was initially immunized with 200 ⁇ g of peptide-KLH mixed in complete Freund's adjuvant.
• the rabbit was then injected every two to three weeks with 200 ⁇ g of peptide-KLH in incomplete Freund's adjuvant. Bleeds were taken approximately 7-10 days following each immunization.
• the titer of the serum was at least 1 :64,000 as determined by ELISA to the peptide.
• Affinity purified 103P2D6 polyclonal antibodies were prepared by passage of crude serum from the immunized rabbit over an affinity matrix comprised of 103P2D6 peptide covalently coupled to Affigel 10 (BioRad). After extensive washing of the matrix with PBS, antibodies specific to 103P2D6 peptide were eluted with low pH glycine buffer (0.1M, pH 2.5).
• PAbs are also prepared by immunization of mice, rabbits, goats, and sheep with recombinant bacterial fusion proteins encoding full length or various regions of the 103P2D6 sequence.
• the recombinant bacterial proteins include glutathione-S-transferase (GST), maltose binding protein (MBP), and HIS tagged fusion proteins of 103P2D6 that are purified from induced bacteria using the appropriate affinity matrix.
• GST glutathione-S-transferase
• MBP maltose binding protein
• HIS tagged fusion proteins of 103P2D6 that are purified from induced bacteria using the appropriate affinity matrix.
• Mammalian expressed secreted Tag5 or FC-fusion proteins encoding the extracellular domain are also used as immunogens for pAb production.
• Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if deshed, an adjuvant.
• the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
• 103P2D6, recombinant bacterial fusion proteins or peptides encoding various regions of the 103P2D6 sequence are used to immunize New Zealand White rabbits.
• a peptide can be designed from a coding region of 103P2D6.
• the peptide can be conjugated to keyhole limpet hemocyanin (KLH) and used to immunize a rabbit.
• KLH keyhole limpet hemocyanin
• the immunizing agent may include all or portions of the 103P2D6 protein, analogs or fusion proteins tliereof.
• the 103P2D6 amino acid sequence can be fused to any one of a variety of fusion protein partners that are well known in the art, such as maltose binding protein, LacZ, thioredoxin or an immunoglobulin constant region (see e.g. Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds, 1995; Linsley, P.S, Brady, W, Urnes, M, Grosmaire, L, Damle, N, and Ledbetter, L.(1991) J. Exp. Med. 174, 561- 566).
• Other recombinant bacterial proteins include glutathione-S-transferase (GST), and HIS tagged fusion proteins of 103P2D6 that are purified from induced bacteria using the appropriate affinity matrix.
• immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
• adjuvants examples include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
• rabbits are initially immunized subcutaneously with about 200 ⁇ g of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant. Rabbits are then injected subcutaneously every two weeks with 200 ⁇ g of immunogen in incomplete Freund's adjuvant. Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.
• the full-length 103P2D6 cDNA can be cloned into an expression vector such as one that provides a 6His tag at the carboxyl-terminus (pCDNA 3.1 myc-his, Invitrogen).
• cell lysates can be probed with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) and the anti-103P2D6 serum using Western blotting.
• specificity of the antiserum is tested by Western blot and immunoprecipitation analyses using lysates of cells that express 103P2D6.
• Serum from rabbits immunized with GST or MBP fusion proteins is first semi-purified by removal of anti-GST or anti-MBP antibodies by passage over GST and MBP protein columns respectively.
• Sera from His- tagged protein and peptide immunized rabbits as well as depleted GST and MBP protein sera are purified by passage over an affinity column composed of the respective immunogen covalently coupled to Afftgel matrix (BioRad).
• 103P2D6 To express 103P2D6 in bacterial cells, portions of 103P2D6 were fused to the glutathione S- fransferase (GST) gene by cloning into pGEX-6P-l (Amersham Pharmacia Biotech, NJ). The constructs were made in order to generate recombinant 103P2D6 protein sequences with GST fused at the N-terminus and a six histidine epitope at the C-terminus. The six histidine epitope tag is generated by adding the histidine codons to the cloning primer at the 3' end of the open reading frame (ORF). A PreScissionTM recognition site permits cleavage of the GST tag from 103P2D6-related protein.
• GST glutathione S- fransferase
• the ampiciUin resistance gene and pBR322 origin permits selection and maintenance of the plasmid in E. coli.
• cDNA encoding the following fragments of 103P2D6 protein were cloned into pGEX-6P-l: amino acids 24 to 487; amino acids 39 to 176; amino acids 170 to 360; and amino acids 1 to 536, or any 8, 9, 10, 11, 12,13, 14 or 15 contiguous amino acids from 103P2D6 or an analog thereof.
• 103P2D6in bacterial cells To express 103P2D6in bacterial cells, all or part of the 103P2D6nucleic acid sequence are fused to the maltose-binding protein (MBP) gene by cloning into pMAL-c2X and pMAL-p2X (New England Biolabs, MA). The constructs are made to generate recombinant 103P2D6protein sequences with MBP fused at the N-terminus and a six histidine epitope at the C-terminus. The six histidine epitope tag is generated by adding the histidine codons to the 3' cloning primer. A Factor Xa recognition site permits cleavage of the GST tag from 103P2D6.
• MBP maltose-binding protein
• the pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.
• cDNA encoding the following fragments of 103P2D6 protein are cloned into pGEX-6P-l: amino acids 24 to 487; amino acids 39 to 176; amino acids 170 to 360; and amino acids 1 to 536, or any 8, 9, 10, 11, 12,13, 14 or 15 contiguous amino acids from 103P2D6 or an analog thereof.
• pCRII construct was generated using cDNA sequence encoding amino acids 44-181.
• the pCRII vector has S ⁇ 6 and T7 promoters flanking the insert to drive the production of 108P5H8 RNA riboprobes which will be used in RNA in situ hybridization experiments.
• the full or partial length 103P2D6cDNA can be cloned into any one of a variety of expression vectors known in the art.
• the constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-103P2D6polyclonal serum, described in Example 4 above, in a Western blot.
• the 103P2D6genes can also be subcloned into the retroviral expression vector pSR ⁇ MSVtkneo and used to establish 103P2D6-expressing cell lines as follows:
• the 103P2D6 coding sequence (from translation initiation ATG and Kozak translation start consensus sequence to the termination codons) is amplified by PCR using ds cDNA template from 103P2D6 cDNA.
• the PCR product is subcloned into pSR ⁇ MSVtkneo vector and transformed into DH5 ⁇ competent cells. Colonies are picked to screen for clones with unique internal restriction sites on the cDNA.
• the positive clone is confirmed by sequencing of the cDNA insert.
• the retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, NIH 3T3, TsuPrl, 293 or rat- 1 cells.
• pcDNA4/HisMax-TOPO Constructs To express 103P2D6 in mammalian cells, the 103P2D6 ORF is cloned into pcDNA4/HisMax-
• the pcDNA4/HisMax-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
• BGH bovine growth hormone
• the Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampiciUin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli.
• the ORF with consensus Kozak translation initiation site is cloned into pcDNA3.1/MycHis_Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter.
• CMV cytomegalovirus
• the recombinant protein has the myc epitope and six histidines fused to the C-terminus to aid in detection and purification of the recombinant protein.
• the pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
• BGH bovine growth hormone
• Neomycrn resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampiciUin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli.
• pcDNA4/V5His-TOPO pcDNA4/V5His-TOPO
• CMV cytomegalovirus
• the recombinant protein has V5TM and six histidine epitopes fused at the C-terminus to aid in detection and purification of the recombinant protein.
• the pcDNA4/V5His-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
• BGH bovine growth hormone
• the Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampiciUin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli.
• the mammalian expression vector pcDNA 3.1 V5-His encoding the 103P2D6 cDNA was used to transfect 293T human embryonic kidney cells to assess 103P2D6 protein expression.
• Western analysis of cell lysates using an anti-103P2D6 pAb reveals an immunoreactive band of approximately 85 kD seen in 293T cells transiently transfected with the 103P2D6 cDNA but not in cells transfected with the control empty vector (FIG. 12A).
• FIG. 12B Western analysis of 103P2D6 mRNA positive HP AC and Bx PC-3 pancreatic cancer cells, CaCo-2 colon cancer cells, and LAPC9 prostate cancer cells, reveals expression of a 60 kD anti-103P2D6 immunoreactive band indicating significant endogenous expression of the 103P2D6 protein (Example 3, above; FIG. 11A-B).
• the increased molecular weight of 103P2D6 in transfected 293T cells may be due to the presence of the V5-His amino acid tag present in the cDNA or post-translational processing or modification of 103P2D6 when overexpressed in 293T cells.
• 103P2D6 protein is expressed on the cell surface of Bx PC-3 cells as indicated by flow cytometric analysis (Example 3, above; FIG.
• pcDNA3.1CT-GFP-TOPO Construct To express 103P2D6 in mammalian cells and to allow detection of the recombinant protein using fluorescence, the ORF with consensus Kozak translation initiation site is cloned into pcDNA3.1CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant protein has the Green Fluorescent Protein (GFP) fused to the C- terminus facilitating non-invasive, in vivo detection and cell biology studies.
• CMV cytomegalovirus
• GFP Green Fluorescent Protein
• the pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen.
• BGH bovine growth hormone
• the Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampiciUin resistance gene and ColEl origin permits selection and maintenance of the plasmid in E. coli.
• An additional construct with a N-terminal GFP fusion is made in ⁇ cDNA3.1NT-GFP-TOPO spanning the entire length of the 103P2D6protein.
• the cDNA encoding 103P2D6 ammo acids 24-487 is cloned mto pAPtag-5 (GenHunter Corp Nashville, TN) This construct generates an alkaline phosphatase fusion at the C-terminus of the 103P2D6prote ⁇ n while fusing the IgGK signal sequence to N-terminus
• the resulting recombmant 103P2D6 protem is optimized for secretion mto the media of transfected mammalian cells and can be used to identify protems such as ligands or receptors that interact with the 103P2D6prote ⁇ n
• Protem expression is driven from the CMV promoter and the recombmant protem also contams myc and six histidines fused to the C-terminus of alkaline phosphatase to aid in detection and purification of the recombmant protem
• the Zeosin resistance gene allows for selection of mammalian cells expressmg the protem
• This vector is similar to pAPtag but without the alkaline phosphatase fusion
• This construct generates an immunoglobulin Gl Fc fusion at the C-terminus of the 103P2D6protem while fusing the IgGK signal sequence to the N-terrmnus
• the resulting recombmant 103P2D6 protem is optimized for secretion mto the media of transfected mammalian cells, and can be used to identify protems such as ligands or receptors that mteract with the 103P2D6prote ⁇ n Protem expression is driven from the CMV promoter and the recombmant protem also contams myc and six histidines fused to the C-termrnus to aid m detection and purification of the re
• the psecFc vector was assembled by cloning immunoglobulin Gl Fc (lunge, CH2, CH3 regions) mto pSecTag2 (Invitrogen, California) This construct geneiates an immunoglobulin Gl Fc fusion at the C-terminus of the 103P2D6protem, while fusing the IgGK signal sequence to N-terrmnus
• the resulting recombmant 103P2D6 protem is optimized for secretion mto the media of transfected mammalian cells, and can be used to identify protems such as ligands or receptors that mteract with the 103P2D6 protem Protem expression is driven from the CMV promoter
• the Zeocm resistance gene allows for selection of mammalian cells that express
• the ORF was cloned into pSR ⁇ constructs.
• Amphotropic and ecotropic retroviruses are generated by transfection of pSR ⁇ constructs into the 293T-10A1 packaging line or co-transfection of pSR ⁇ and a helper plasmid ( ⁇ ) in the 293 cells, respectively.
• the refrovirus can be used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 103P2D6, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR).
• LTR long terminal repeat
• Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampiciUin resistance gene and ColEl origin permit selection and maintenance of the plasmid in E. coli.
• An additional pSR ⁇ construct was made that fused the FLAG tag to the C-terminus to allow detection using anti-FLAG antibodies.
• the FLAG sequence 5' gat tac aag gat gac gac gat aag 3' was added to cloning primer at the 3' end of the ORF.
• Additional pSR ⁇ constructs are made to produce both N-terminal and C-terminal GFP and myc/6 HIS fusion proteins of the full-length 103P2D6protein.
• Example 6 Production of Recombinant 103P2D6in a Baculovirus System
• cDNA sequence encoding the 103P2D6 protehi is cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus Specifically, pBlueBac ⁇ 103P2D6 is co- transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
• pBlueBac 4.5 Invitrogen
• Recombinant 103P2D6protein is then generated by infection of HighFive insect cells (Invitrogen) with the purified baculovirus.
• Recombinant 103P2D6 protein can be detected using anti- 103P2D6 antibody.
• 103P2D6 protein can be purified and used in various ceU-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 103P2D6.
• Example 7 Chromosomal Mapping of the 103P2D6 Gene The chromosomal localization of 103P2D6 was determined using the GeneBridge4
• PCR primers were used to localize 103P2D6: 103P2D6.1 cttgggaggtcctagtgctaagtg
• mapping vector for the 93 radiation hybrid panel DNAs was: 0100011101011010011011111100112001001000010011100111110010001011011001001101101101 10101000011.
• This vector and the mapping program at http://www-genome.wi.mit.edu cgi- bin/contig/rhmapper.pl placed 103P2D6 on chromosome 4pl2-pl4 (D4S3197-D4S1577).
• Example 8 Identification of Potential Signal Transduction Pathways Based on its surface localization, 103P2D6 can regulate key signaling pathways.
• 103P2D6 Several pathways known to play a role in cancer biology can be regulated by 103P2D6, including phospholipid pathways such as PI3K, AKT, etc, as well as mitogenic/survival cascades such as ERK, ⁇ 38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003.).
• the role that 103P2D6 plays in the regulation of these pathways can be investigated using, Western blotting techniques and reporter assays.
• Cells lacking 103P2D6 and cells expressing 103P2D6 are either left untreated or stimulated with serum, cytokines, androgen or antibodies.
• Cell lysates are analyzed using anti-phosphos-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and other signaling molecules.
• 103P2D6 plays a role in the regulation of signaling pathways, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• luciferase (luc) based transcriptional reporter assays are carried out in cells that express 103P2D6. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. Some of the reporters and examples of these associated ttanscription factors, signal transduction pathways, and activation stimuli are listed below.
• NFkB-luc NFkB/Rel
• Ik-kinase/SAPK growth/apoptosis/stress
• CRE-luc, CREB/ATF2 PKA/p38; growth/apoptosis/stress
• 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• 103P2D6-mediated effects are assayed in cells showing 103P2D6 mRNA expression.
• Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer. This assay allows one to determine the effect of signaling pathways activation.
• 103P2D6 plays a role in activation signaling pathways, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• Example 9 Generation of 103P2D6 Monoclonal Antibodies (mAbsl To generate mAbs to 103P2D6, mice are immunized inttaperitoneally with 10-50 ⁇ g of protein immunogen mixed in complete Freund's adjuvant. Protein immunogens include peptides, recombinant 103P2D6 proteins, and, mammalian expressed human IgG FC fusion proteins. Mice are then subsequently immunized every 2-4 weeks with 10-50 ⁇ g of antigen mixed in Freund's incomplete adjuvant. Alternatively, Ribi adjuvant is used for initial immunizations.
• a DNA-based immunization protocol in which a mammalian expression vector used to immunize mice by direct injection of the plasmid DNA.
• a mammalian expression vector used to immunize mice by direct injection of the plasmid DNA For example, a pCDNA 3.1 encoding 103P2D6 cDNA alone or as an IgG FC fusion is used. This protocol is used alone or in combination with protein immunogens. Test bleeds are taken 7-10 days following immunization to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, and immunoprecipitation analyses, fusion and hybridoma generation is then carried with established procedures well known in the art (Harlow and Lane, 1988).
• a glutathione-S- transferase (GST) fusion protein encompassing a 103P2D6 protein is synthesized and used as immunogen.
• GST glutathione-S- transferase
• Balb C mice are initially immunized intraperitoneally with 200 ⁇ g of the GST-103P2D6 fusion protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 75 ⁇ g of GST-103P2D6 protein mixed in Freund's incomplete adjuvant for a total of three immunizations.
• Reactivity of serum from immunized mice to full-length 103P2D6 protein is monitored by ELISA using a partially purified preparation of HIS-tagged 103P2D6 protein expressed from 293T cells (Example 5). Mice showing the strongest reactivity are rested for three weeks and given a final injection of fusion protein in PBS and then sacrificed four days later. The spleens of the sacrificed mice are then harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from growth wells following HAT selection are screened by ELISA and Western blot to identify 103P2D6 specific antibody-producing clones.
• the binding affinity of a 103P2D6 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and can be used to help define which 103P2D6 monoclonal antibodies are preferred for diagnostic or therapeutic use.
• the BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity.
• the BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
• 103P2D6 contributes to the growth of prostate and other cancer cells (See Table I) by several mechanisms.
• the 103P2D6 protein can function as a cell surface receptor or as a transporter and contribute to tumor growth by regulating tumor cell proliferation or responding to tumor cells, endothelial cells or steoma. Alternatively, 103P2D6 contributes to tumor growth by mediating cellular adhesion, transformation or downstream gene expression.
• the functions of 103P2D6 can be evaluated, e.g, by using engineered cell lines that express 103P2D6. For example, primary cells such as PrEC, cancer cell lines and NIH 3T3 cells engineered to stably express 103P2D6 are evaluated for cell growth potential. When 103P2D6 participates in neoplastic cell growth, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• 103P2D6 plays in transformation is evaluated.
• Primary cells such as PrEC, and NIH3T3 cells engineered to express 103P2D6 are compared to 103P2D6-negative cells, to evaluate their ability to form colonies in soft agar (Song Z. et al. Cancer Res. 2000; 60:6730), where colony formation indicates the presence of transformed cells.
• 103P2D6 mediates transformation
• 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• 103P2D6 in the various cancers listed in Table I indicates that this gene has a functional role in tumor progression.
• 103P2D6 function can be assessed in mammalian cells using in vitro approaches. Mammalian cells infected with the retroviral vector pSR ⁇ tkneo or pSR ⁇ -103P2D6 are compared (Muller et al, 1991, MCB 11:1785) using a variety of assays, including cell proliferation in tissue culture and in vitro invasion using a membrane invasion culture system (Welch et al. Int. J. Cancer 43: 449-457).
• Cell lines expressing 103P2D6 are assayed for invasive and migratory properties by measuring passage of cells through a mattigel coated TranswellTM system (Becton Dickinson) (Cancer Res. 1999; 59:6010). Passage of cells through the membrane to the opposite side is monitored using a fluorescent assay (Becton Dickinson Technical Bulletin #428). For example, cells lacking 103P2D6 and cells expressing 103P2D6 are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population. When 103P2D6 is involved with cell invasion, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• Envelope proteins have been shown to exhibit chemotactic abilities (Lin CL, Sewell AK, Gao fGF, Whelan KT, Phillips RE, Austyn JM. J Exp Med. 2000, 192:587).
• indicator cells are monitored for passage through the Transwell system toward a gradient of 103P2D6-conditioned media compared to control media.
• This assay can also be used to qualify and quantify specific neutralization of 103P2D6 effects.
• the neutralization can be effected by candidate cancer therapeutic compositions.
• 103P2D6 When 103P2D6 mediates tissue invasion, such as by chemotaction, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• the function of 103P2D6 can be evaluated using anti-sense RNA technology coupled to the various functional assays described herein, e.g. growth, invasion and migration.
• Anti-sense RNA oligonucleotides can be introduced into 103P2D6 expressing cells, thereby preventing the expression of 103P2D6.
• Control and anti-sense containing cells can be analyzed for proliferation, invasion, migration, apoptotic and transcriptional potential. The local as well as systemic effect of the loss of 103P2D6 expression can be evaluated.
• Envelope proteins have been shown to mediate cell adhesion and syncytium formation (J. Immunol. 1996, 156:1307; AIDS. 1991, 5:1425). Based on its similarity with envelope proteins, 103P2D6 can mediate protein-protein association resulting in multimeric complexes. The association of proteins into large complexes is critical in several biological processes, including signal transduction, cell communication, ubiquitination, transcriptional regulation, etc.
• 103P2D6 can participate in regulating cell adhesion and communication.
• cells lacking 103P2D6 are compared to cells expressing 103P2D6, using techniques known in the art (see, e.g, Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118).
• a fluorescent indicator such as calcein
• Adherent cells are detected by fluorimettic analysis.
• Example 12 In Vivo Assay for 103P2D6 Tumor Growth Promotion
• the effect of the 103P2D6 protein on tumor cell growth can be evaluated in vivo by gene overexpression in tumor-bearing mice.
• SOD mice can be injected SQ on each flank with 1 x 10 6 of either PC3, TSUPR1, or DU145 cells containing tkNeo empty vector or 103P2D6.
• Constitutive 103P2D6 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a refrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems.
• a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus,
• Regulated expression under control of an inducible vector system such as ecdysone, tet, etc, can be used provided such promoters are compatible with the host cell systems.
• Tumor volume is then monitored at the appearance of palpable tumors and is followed over time to determine if 103P2D6-expressing cells grow at a faster rate and whether tumors produced by 103P2D6-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
• mice can be implanted with 1 x 10 5 of the same cells orthotopically to determine if 103P2D6 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow. Also see saffron et al, "Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts" PNAS (in press, 2001).
• the assay is also useful to determine the 103P2D6 inhibitory effect of candidate therapeutic compositions, such as for example, 103P2D6 intrabodies, 103P2D6 antisense molecules and ribozymes.
• 103P2D6 The cellular location of 103P2D6 can be assessed using subcellular fractionation techniques widely used in cellular biology (Storrie B, et al. Methods Enzymol. 1990; 182:203-25). Prostate or other cell lines can be separated into nuclear, cytosolic and membrane fractions. The expression of 103P2D6 in the different fractions can be tested using Western blotting techniques.
• 293T cells can be transfected with an expression vector encoding HIS-tagged 103P2D6 (PCDNA 3.1 MYC/HIS, Invitrogen).
• the transfected cells can be harvested and subjected to a differential subcellular fractionation protocol as previously described (Pemberton, PA. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.) This protocol separates the cell into fractions enriched for nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble proteins.
• 103P2D6 is understood to be associated with the cell surface.
• Surface staining experiments confirm that 103P2D6 cDNA is expressed at the cell surface (see, e.g. Figures 11- 12).
• the potential functions of 103P2D6 include (1) a surface receptor that transmits signals to the cell nucleus, or (2) a ttansporter that moves ions and proteins from in and out of the cell, or (3) a mediator of cell adhesion and cell-cell interaction.
• the cellular location of 103P2D6 can be assessed using subcellular fractionation techniques widely used in cellular biology (see, e.g, Storrie B, et al. Methods Enzymol. 1990; 182:203-25).
• Prostate, colon, bladder, kidney or pancreas tumor cell lines are separated into nuclear, cytosolic, heavy membranes (lysosomes, peroxisomes, and mitochondria) and light membranes (plasma membrane and endoplasmic reticulum) fractions.
• the expression of 103P2D6 is followed in each fraction by Western blotting.
• 103P2D6 participates in cell adhesion or cell-cell communication, 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• 103P2D6 determines the specific proteins with which 103P2D6 associates, including cytoskeleton and integrins, can be made, e.g, using co-precipitation and Western blotting techniques (see, e.g, Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261:646). Immunoprecipitates from cells expressing 103P2D6 and cells lacking 103P2D6 are compared for specific protein-protein associations. 103P2D6 also associates with effector molecules, such as C2- domain containing proteins.
• the 103P2D6 protein contains a leucine zipper at its ammo-terminus.
• Leucine zippers play a role in protein dimerization and determine sequence-specific DNA binding (Luscher B, Larsson LG. Oncogene. 1999; 18:2955), and are therefore transcriptional regulators.
• 103P2D6 can regulate tumor progression by regulating gene expression.
• the role that 103P2D6 plays in regulating gene expression can be evaluated, e.g, by studying gene expression in cells expressing or lacking 103P2D6. For example, RNA from parental and 103P2D6-expressing NIH3T3 and PC3 cells is extracted and hybridized to commercially available gene arrays (Clontech).
• 103P2D6 regulates transcription
• 103P2D6 is used as a target for diagnostic, preventative and therapeutic purposes.
• Example 17 103P2D6 monoclonal antibody mediated inhibition of prostate tumors in vivo
• the significant expression of 103P2D6, in cancer tissues, together with its restrictive expression in normal tissues makes 103P2D6 a target for antibody therapy.
• 103P2D6 is a target for T cell-based immunotherapy.
• the therapeutic efficacy of anti-103P2D6 mAbs in human prostate cancer xenograft mouse models is evaluated by using the androgen-dependent LAPC-9 xenograft (Craft, N, et al, Cancer Res, 1999. 59(19): p. 5030-6) and the androgen independent recombinant cell line PC3-103P2D6 (see, e.g, Kaighn, M.E, et al, Invest Urol, 1979. 17(1): p. 16-23).
• Antibody efficacy on tumor growth and metastasis formation is studied, e.g, in a mouse orfhotopic prostate cancer xenograft model. These studies demonstrate that anti-103P2D6 MAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-rndependent PC3-103P2D6 tumor xenografts. Anti-103P2D6 mAbs also retard the growth of established orfhotopic tumors significantly and prolonged survival of tumor-bearing mice. These results indicate the utility of anti- 103P2D6 mAbs in the treatment of local and advanced stages of prostate cancer.
• Monoclonal antibodies are raised against 103P2D6 as described in Example 9.
• Antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 103P2D6.
• Epitope mapping data for the anti-103P2D6 mAbs as determined by ELISA and Western analysis, recognize epitopes on the 103P2D6 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.
• Antibody Formulation The monoclonal antibodies are purified from hybridoma tissue culture supernatants by Protein-
• a therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orfhotopic injections of LAPC-9 prostate tumor xenografts.
• the LAPC-9 xenograft which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SOD) mice (Taconic Farms) by s.c. trocar implant (Craft, N, et al, supra). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al.
• the prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% (vol vol) FBS.
• a PC3-103P2D6 cell population is generated by retroviral gene transfer as described in Hubert, R.S, et al, Proc Natl Acad Sci U S A, 1999. 96(25): p. 14523-8.
• Anti-103P2D6 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.
• Subcutaneous (s.c.) tumors are generated by injection of 1 x 10 6 LAPC-9, PC3, or PC3- 103P2D6 cells mixed at a 1:1 dilution with Mattigel (Collaborative Research) in the right flank of male SOD mice.
• To test antibody efficacy on tumor formation i.p. antibody injections are started on the same day as tumor-cell injections.
• mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth.
• Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length x width x height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of anti-103P2D6 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). Orfhotopic injections are performed under anesthesia by using ketamine/xylazine. An incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate.
• LAPC-9 cells (5 x 10 5 ) mixed with Matrigel are injected into each dorsal lobe in a 10- ⁇ l volume.
• mice are bled on a weekly basis for determination of PSA levels. Based on the PSA levels, the mice are segregated into groups for the appropriate treatments.
• anti-103P2D6 mAbs are started when PSA levels reach 2-80 ng/ l.
• LAPC-9 tumor cells are injected into the mouse prostate, and 2 days later, the mice are segregated into two groups and treated with either 200 ⁇ g of 1G8 mAb or PBS three times per week for two weeks. Mice are monitored weekly for circulating PSA levels as an indicator of tumor growth.
• a major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by THC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R.S, et al, Proc Natl Acad Sci U S A, 1999. 96(25): p. 14523-8).
• mice bearing established orthotopic LAPC-9 tumors are administered 11 injections of either anti-103P2D6 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate and lungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHC analysis.
• Anti-103P2D6 antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-103P2D6 mAbs demonsfrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-103P2D6 mAbs are efficacious on major clinically relevant end points/PSA levels (tumor growth), prolongation of survival, and health.
• Phosphorylation whether by PKC, cAMP and c-GMP dependent kinases, or casern kinases, exhorts a profound effect on proteins. Phosphorylation mediates protein-protein interactions as well as signaling pathway activation. Phosphorylation also controls protein localization, translocation and enzymatic activity, and regulates transcriptional activation (Mol Immunol. 2000, 37: 1; Cell Mol Life Sci. 2000, 57:1172-83). By means of post-translational modification of protein, phosphorylation regulates cellular functions, including proliferation, migration and gene expression (Cell Prolif. 2000, 33:341; Mol Biol Cell. 2001, 12:351).
• Myristoylation serves to anchor numerous proteins to the cytoplasmic face of the plasma membrane. This process serves to facilitate protein recruitment, complex assembly and signaling through proteins (Current Opinion Cell Biol. 1994, 6:219; J Biol Chem. 1996, 271:1573).
• 103P2D6 is proposed to be a membrane associated protein, primarily expressed at the cell surface (64%). There is a possibility that 103P2D6 is associated with the endoplasmic reticulum (21%).
• PSORT http://psort.nibb.ac.ip indicates the presence of 1 TM domain at aa 493-509 (VLLIVLFCLFIFVLIYV), and 1 signal sequence at aa 1-24 (MGSLSNCALLQLTLTAFLTILVQP), with a cleavage site between aa 24 and aa 25.
• TMpred www.ch.3mbnet.org indicates that 103P2D6 may have four transmembrane domains, which are listed below: TM1 aa 4-22 - o-i
• the N-terminus extends outside the cell, while the C-terminus is intracellular.

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